Combination of Bcl-2/Bcl-xL inhibitors and chemotherapeutic agent and use thereof

ABSTRACT

The present disclosure provides a pharmaceutical composition comprising a Bcl-2/Bcl-xL inhibitor, a chemotherapeutic agent, and a pharmaceutically acceptable carrier. The present disclosure also provides a method of treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a Bcl-2/Bcl-xL inhibitor alone or in combination with a therapeutically effective amount of a chemotherapeutic agent. The present disclosure also provides use of a combination of a Bcl-2/Bcl-xL inhibitor and a chemotherapeutic agent in the manufacture of an anti-tumor medicament. In the present disclosure, a significantly enhanced anti-tumor effect can be achieved by administration of a Bcl-2/Bcl-xL inhibitor in combination with a chemotherapeutic agent.

RELATED APPLICATIONS

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 16/630,855, filed on Jan. 14, 2020, which claimspriority to Chinese patent application No. 201810914839.4, filed on Jul.31, 2018, PCT application No. PCT/CN2019/074862, filed on Feb. 12, 2019,Chinese patent application No. 201910598720.5, filed on Jul. 4, 2019,and Chinese patent application No. 201910662265.0, filed on Jul. 22,2019, the contents of each of which are hereby incorporated by referencein their entirety.

FIELD OF THE INVENTION

The present disclosure relates to a pharmaceutical composition and usethereof in the treatment of cancer, and a method of treating cancercomprising a Bcl-2/Bcl-xL inhibitor and a chemotherapeutic agent.

BACKGROUND

Apoptosis is a natural pathway for the body to clear abnormal orunwanted cells, and if it is affected, it may lead to various diseasessuch as cancer. Bcl-2 family proteins are important regulators ofapoptosis. The family of proteins includes anti-apoptotic proteins suchas Bcl-2, Bcl-xL and Mcl-1; and pro-apoptotic molecules, including Bid,Bim, Bad, Bak and Bax. Although normal cells have low expression levelsof anti-apoptotic Bcl-2 and Bcl-xL proteins, these proteins are found tobe highly overexpressed in many different types of human tumors and areimplied in tumor development, progression and resistance to drugs.Targeting Bcl-2 and/or Bcl-xL has been investigated as a cancertreatment strategy.

The first generation Bcl-2/Bcl-xL inhibitors ABT-737 and ABT-263 aresmall molecule Bcl-2 inhibitors developed by Abbott Laboratories, USA.ABT-737 and ABT-263 have been reported to bind Bcl-2, Bcl-xL and Bcl-w(Ki<1 nM) with high affinity, and the binding has a higher specificitythan their binding to Mcl-1 and A1 (two other anti-apoptotic Bcl-2proteins). In addition, a second generation Bcl-2/Bcl-xL inhibitor hasbeen disclosed in WO 2014/113413 A1, which binds to Bcl-2 and/or Bcl-xL,with a Ki value of <10 nM, and functions as a potent antagonist of Bcl-2and Bcl-xL in cell-free function assays.

Combination therapy of anti-cancer drugs is sometimes used in thetreatment of cancer. There exists needs for a combination of aBcl-2/Bcl-xL inhibitor having high anti-cancer activity and low sideeffects and other anti-cancer agents.

SUMMARY OF INVENTION

It is an intent of the present disclosure to provide a pharmaceuticalcomposition having a notable anti-cancer effect, a method for treatingcancer, and use of the pharmaceutical composition.

According to a first aspect of the present disclosure, provided hereinis a pharmaceutical composition comprising a Bcl-2/Bcl-xL inhibitor anda chemotherapeutic agent, and a pharmaceutically acceptable carrier. TheBcl-2/Bcl-xL inhibitor is a compound of the following formula (I), (II),(III) or (IV), or a pharmaceutically acceptable salt thereof:

wherein the A ring is

X, substituted or unsubstituted, is selected from the group consistingof alkylene, alkenylene, cycloalkylene, cycloalkenylene, andheterocycloalkylene;Y is selected from the up consisting of (CH₂)_(n)—N(R^(a))₂ and

Q is selected from the group consisting of O, O(CH₂)₁₋₃, NR^(c),NR^(c)(C₁₋₃alkylene), OC(═O)(C₁₋₃alkylene), C(═O)O,C(═O)O(C₁₋₃alkylene), NHC(═O)(C₁₋₃alkylene), C(═O)NH, andC(═O)NH(C₁₋₃alkylene);Z is O or NR^(c);R₁ and R₂, independently, are selected from the group consisting of H,CN, NO₂, halo, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl,heteroaryl, heterocycloalkyl, OR′, SR′, NR′R″, COR′, CO₂R′, OCOR′,CONR′R″, CONR′SO₂R″, NR′COR″, NR′CONR″R′″, NR′C═SNR″R′″, NR′SO₂R″,SO₂R′, and SO₂NR′R″;R₃ is selected from a group consisting of H, alkyl, cycloalkyl, alkenyl,cycloalkenyl, alkynyl, aryl, heteroaryl, heterocycloalkyl, OR′, NR′R″,OCOR′, CO₂R′, COR′, CONR′R″, CONR′SO₂R″, C₁₋₃alkyleneCH(OH)CH₂OH, SO₂R′,and SO₂NR′R″;R′, R″, and R′″, independently, are H, alkyl, cycloalkyl, alkenyl,cycloalkenyl, alkynyl, aryl, heteroaryl, C₁₋₃alkyleneheterocycloalkyl,or heterocycloalkyl;R′ and R″, or R″ and R′″, can be taken together with the atom to whichthey are bound to form a 3 to 7 membered ring;R₄ is hydrogen, halo, C₁₋₃alkyl, CF₃, or CN;R₅ is hydrogen, halo, C₁₋₃alkyl, substituted C₁₋₃alkyl, hydroxyalkyl,alkoxy, or substituted alkoxy;R₆ is selected from the group consisting of H, CN, NO₂, halo, alkyl,cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl,heterocycloalkyl, OR′, SR′, NR′R″, CO₂R′, OCOR′, CONR′R″, CONR′SO₂R″,NR′COR″, NR′CONR″R′″, NR′C═SNR″R′″, NR′SO₂R″, SO₂R′, and SO₂NR′R″;R₇, substituted or unsubstituted, is selected form the group consistingof hydrogen, alkyl, alkenyl, (CH₂)₀₋₃cycloalkyl, (CH₂)₀₋₃cycloalkenyl,(CH₂)₀₋₃heterocycloalkyl, (CH₂)₀₋₃aryl, and (CH₂)₀₋₃heteroaryl;R₈ is selected form the group consisting of hydrogen, halo, NO₂, CN,CF₃SO₂, and CF₃;R_(a) is selected from the group consisting of hydrogen, alkyl,heteroalkyl, alkenyl, hydroxyalkyl, alkoxy, substituted alkoxy,cycloalkyl, cycloalkenyl, and heterocycloalkyl;R_(b) is hydrogen or alkyl;R_(c) is selected from the group consisting of hydrogen, alkyl,substituted alkyl, hydroxyalkyl, alkoxy, and substituted alkoxy;n, r, and s, independently, are 1, 2, 3, 4, 5, or 6;R₂₁ is SO₂R₂′,R₂₂ is alkyl, preferably C₁₋₄ alkyl, more preferably methyl, propyl, orisopropyl,R₂₃ is alkyl, preferably C₁₋₄ alkyl, more preferably methyl, propyl, orisopropyl,R₂₄ is halogen, preferably fluoride, chloride,R₂₅ is halogen, preferably fluoride, chloride,R₂₆ is selected from H, halogen, alkyl, preferably fluoride, chloride,C₁₋₄ alkyl, more preferably methyl, propyl, isopropylR_(21b) is H or alkyl, preferably C₁₋₄ alkyl, more preferably methyl,propyl, or isopropyl,n₂, r₂ and s₂ are independently 1, 2, 3, 4, 5 or 6, more preferably, r₂and s₂ are both 2 and n₂ is 3, 4 or 5, more preferably, all of n₂, r₂and s₂ are 2; andR₂′ is alkyl, preferably C₁₋₄ alkyl, more preferably methyl, propyl, orisopropyl.

The pharmaceutical composition of the present disclosure has anexcellent anti-cancer effect, and in particular, the anti-cancer effectof the combination of the Bcl-2/Bcl-xL inhibitor and thechemotherapeutic agent of the present disclosure is superior to the sumof the therapeutic effects of the respective individual components,i.e., the pharmaceutical composition of the present disclosure achievesan excellent synergistic effect.

Compounds of the above formula (I), (II) or (III) have been disclosed inWO 2014/113413 A1, which is incorporated herein by reference to itsentirety. The above formula (IV) is disclosed in PCT/CN2019/070508,which is incorporated herein by reference.

In some embodiments, Y is Y is

n is an integer of 1, 2 or 3;R_(b) is hydrogen or C₁₋₃alkyl;Q is O, O(CH₂)₁₋₃, C(═O)O(CH₂)₁₋₃, OC(═O)(CH₂)₁₋₃, or C(═O)O(C₃H₇)₁₋₃.

In some embodiments, the Bcl-2/Bcl-xL inhibitor is selected from a groupconsisting of the following compounds:

In some embodiments, the Bcl-2/Bcl-xL inhibitor is

(R)-2-(1-(3-(4-N-(4-(4-(3-(2-(4-chlorophenyl)-1-isopropyl-5-methyl-4-(methylsulfonyl)-1H-pyrrol-3-yl)-5-fluorophenyl)piperazin-1-yl)phenyl)sulfamoyl)-2-(trifluoromethylsulfonyl)phenylamino)-4-(phenylthio)butyl)piperidine-4-carbonyloxy)ethylphosphonicacid of the following formula, or a pharmaceutically acceptable saltthereof:

or(R)-1-(3-(4-(N-(4-(4-(3-(2-(4-chlorophenyl)-1-isopropyl-5-methyl-4-(methylsulfonyl)-1H-pyrrol-3-yl)-5-fluorophenyl)piperazin-1-yl)phenyl)sulfamoyl)-2-(trifluoromethylsulfonyl)phenylamino)-4-(phenylthio)butyl)piperidine-4-carboxylicacid of the following formula, or a pharmaceutically acceptable saltthereof:

In some embodiments, the chemotherapeutic drug is selected from thegroup consisting of panobinostat, actinomycin, all-trans retinoic acid,azacitidine, azathioprine, bleomycin, bortezomib, carboplatin,capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytosinearabinoside, daunorubicin, docetaxel, 5-fluorouracil,deoxyfluorouridine, doxorubicin, epirubicin, adriamycin, epothilone,etoposide, fluorouracil, gemcitabine, hydroxyurea, idarubicin, imatinib,irinotecan, nitrogen mustard, Mercaptopurine, methotrexate,mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, teniposide,thioguanine, topotecan, valrubicin, vemurafenib, vinblastine,vincristine, vindesine, vinorelbine and hydroxycamptothecin.

In some embodiments, the chemotherapeutic drug is selected from thegroup consisting of docetaxel, panobinostat, 5-fluorouracil, paclitaxel,cisplatin, irinotecan, topotecan, and etoposide.

In some embodiments, the pharmaceutical composition is docetaxel,panobinostat or 5-fluorouracil.

In some embodiments, the pharmaceutical composition is for use in thetreatment of cancer.

According to another aspect of the invention, provided herein is amethod of treating cancer comprising administering a therapeuticallyeffective amount of a Bcl-2/Bcl-xL inhibitor and a therapeuticallyeffective amount of a chemotherapeutic agent to an individual in needthereof. The Bcl-2/Bcl-xL inhibitor and the chemotherapeutic agent areas defined in the present invention.

According to another aspect of the invention, provided herein is amethod of treating a cancer in a patient in need of, comprisingadministering to the subject a Bcl-2/Bcl-xL inhibitor twice or once aweek at a dose from about 10 mg to about 400 mg, wherein theBcl-2/Bcl-xL inhibitor is as defined in the present invention.

In some embodiments, the cancer is selected from the group consisting ofadrenal cancer, lymphoepithelial neoplasia, adenoid cell carcinoma,lymphoma, acoustic neuroma, acute lymphocytic leukemia, acrallentiginous melanoma, acute myeloid leukemia, acrospiroma, chroniclymphocytic leukemia, acute eosinophilic leukemia, liver cancer, acuteerythrocyte leukemia, small cell lung cancer, acute lymphocyticleukemia, non-small cell lung cancer, acute megakaryoblastic leukemia,MALT lymphoma, acute monocytic leukemia, malignant fibrous histiocytoma,acute promyelocytic leukemia, malignant peripheral schwannomas,adenocarcinoma, mantle cell lymphoma, adenocarcinoma, marginal zoneB-cell lymphoma, malignant hippocampal tumor, adenoid cystic carcinoma,gland tumor, adenoma-like odontogenic tumor, mast cell leukemia,adenosquamous carcinoma, mediastinal germ cell tumor, adipose tissuetumor, breast medullary carcinoma, adrenocortical carcinoma, medullarythyroid carcinoma, adult T cell leukemia/lymphoma, Medulloblastoma,invasive NK cell leukemia, melanoma, AIDS-related lymphoma, meningioma,lung rhabdomyosarcoma, Merkel cell carcinoma, alveolar soft tissuesarcoma, mesothelioma, ameloblastoma, metastatic urothelial carcinoma,anaplastic large cell lymphoma, mixed Müllerian tumor, thyroidundifferentiated carcinoma, mucinous neoplasm, angioimmunoblastic T-celllymphoma, multiple myeloma, angiomyolipoma, muscle tissue tumor,angiosarcoma, mycosis fungoides, astrocytoma, myxoid liposarcoma,atypical deformed rhabdoid tumor, myxoma, B-cell chronic lymphocyticleukemia, mucinous sarcoma, B-cell lymphoblastic leukemia,nasopharyngeal carcinoma, B-cell lymphoma, schwannomas, basal cellcarcinoma, neuroblastoma, biliary tract cancer, neurofibromatosis,bladder cancer, neuroma, blastoma, nodular melanoma, bone cancer, eyecancer, Brenner tumor, oligodendroxoma, brown tumor, oligodendroglioma,Burkitt's lymphoma, eosinophilic breast cancer, brain cancer, opticnerve tumor cancer, oral cancer carcinoma in situ, osteosarcoma,carcinosarcoma, ovarian cancer, cartilage tumor, pulmonary sulcus tumor,papillary thyroid carcinoma, myeloma, paraganglioma, chondroma, pinealblastoma, chordoma, pineal cell tumor, choriocarcinoma, pituitary tumor,choroid plexus papilloma, pituitary adenoma, kidney clear cell sarcoma,pituitary tumor, craniopharyngioma, plasmacytoma, cutaneous T-celllymphoma, multiple embryonic cell tumor, cervical cancer, precursor Tlymphoblastic lymphoma, colorectal cancer, primary central nervoussystem lymphoma, Degos disease, primary effusion lymphoma, proliferativesmall round cell tumor, primary preformed peritoneal cancer, prostatecancer, embryonic dysplasia of neuroepithelial neoplasia, pancreaticcancer, anaplastic cell tumor, pharyngeal carcinoma, embryoniccarcinoma, peritoneal pseudomyxoma, endocrine gland tumor, renal cellcarcinoma, enteropathy-associated T-cell lymphoma, endodermal sinustumor, renal medullary carcinoma, retinoblastoma, esophageal cancer,rhabdomyosarcoma, endadelphos, rhabdomyosarcoma, fibroids, Richter'stransformation, fibrosarcoma, rectal cancer, follicular lymphoma,sarcoma, follicular thyroid cancer, schwannoma, ganglion cell tumor,seminoma, gastrointestinal cancer, Sertoli cell tum, germ cell tumor,sex cord-gonadal stromal tumor, pregnancy choriocarcinoma, signet ringcell carcinoma, giant cell fibroblastoma, skin cancer, bone giant celltumor of bone, small blue round cell tumor, glioma, small cellcarcinoma, glioblastoma multiforme, soft tissue sarcoma, glioma,somatostatin tumor, glioma brain, soot wart, pancreatic highglucagonoma, spinal tumor, Gonadoblastoma, spleen marginal lymphoma,granulosa cell tumor, squamous cell carcinoma, estrogen tumor, synovialsarcoma, gallbladder cancer, Sezary disease, gastric cancer, smallintestine cancer, hairy cell leukemia, squamous cell carcinoma,hemangioblastoma, gastric cancer, head and neck cancer, T-cell lymphoma,vascular epithelioma, testicular cancer, hematological malignancies,sarcoma, hepatoblastoma, thyroid cancer, hepatosplenic T-cell lymphoma,transitional cell carcinoma, Hodgkin's lymphoma, laryngeal cancer,non-Hodgkin's lymphoma, urachal cancer, invasive lobular carcinoma,genitourinary cancer, intestinal cancer, urothelial carcinoma, renalcancer, uveal melanoma, laryngeal cancer, uterine cancer, malignantfreckle-like sputum, verrucous carcinoma, lethal midline granuloma,visual pathway glioma, leukemia, vulvar cancer, testicular stromaltumor, vaginal cancer, liposarcoma, Waldenstrom's macroglobulinemiaDisease, lung cancer, adenolymphoma lymphangioma, nephroblastoma,lymphangisarcoma.

In some embodiments, the cancer is selected from the group consisting ofbreast cancer (e.g., triple negative breast cancer), lung cancer (e.g.,small cell lung cancer or non-small cell lung cancer), prostate cancer,gastric cancer and colon cancer.

In some embodiments, the cancer is selected from the group consisting ofsmall cell lung cancer (SCLC), prostate cancer (e.g., prostateadenocarcinoma), ovarian cancer (e.g., epithelial ovarian tumor, ovariangerm cell tumor, ovarian stromal tumor), gastric cancer (e.g., gastricadenocarcinoma), pancreatic cancer (e.g., exocrine cancers, or ampullarycancer), atypical thymoma, breast cancer (e.g., ductal carcinoma insitu, invasive breast carcinoma, phyllodes tumors, or breastangiosarcoma), non-small cell lung cancer (NSCLC, e.g., lungadenocarcinoma, squamous cell lung carcinoma, or large cell lungcarcinoma), melanoma, colon cancer and brain cancer (e.g., glioma,astrocytoma, oligodendroglioma, ependymoma, meningioma, medulloblastoma,ganglioglioma, Schwannoma, or craniopharyngioma).

In some embodiments, the cancer is a solid tumor.

In some embodiments, the cancer is a metastatic solid tumor.

In some embodiments, the cancer is Bcl-2/Bcl-xL and Bax positive.

In some embodiments, the cancer is gastric cancer.

In some embodiments, the cancer is resistant to an EGFR inhibitor.

In some embodiments, the cancer is lung cancer (e.g., small cell lungcancer or non-small cell lung cancer).

In some embodiments, the EGFR inhibitor is selected from the groupconsisting of gefitinib, erlotinib, ectinib, afatinib, dacomitinib,imatinib, lapatinib, osimertinib (AZD9291), nazatinib, rociletinib,naquotinib, vandetanib, neratinib, pelitinib, canertinib, briatininib,PKC412, Go6976, mavelertinib, olmutinib, WZ4002, TAS2913, cetuximab,panitumumab, avitinib, HS-10296 and TQB3804.

In some embodiments, the cancer is characterized by expressing a mutatedEGFR having one or more mutations selected from the group consisting ofL858R, T790M, C797S, and EGFR gene exon 20 insertion.

In some embodiments, the EGFR has the following mutations: L858R, T790M,and C797S.

In some embodiments, the patient is diagnosed as expressing the mutantEGFR.

In some embodiments, the patient is relapsed from or refractory to aprior treatment.

In some embodiments, the prior treatment comprises surgery,chemotherapy, radio therapy, targeted therapy, immunotherapy, or anycombination thereof.

In some embodiments, the Bcl-2/Bcl-xL inhibitor is administered at adose of about 10 mg, 20 mg, 40 mg, 80 mg, 160 mg, 240 mg, 320 mg, or 400mg.

In some embodiments, the treatment comprises one or more 28-daytreatment cycle.

In some embodiments, the the treatment comprises administering theBcl-2/Bcl-xL inhibitor on days 1, 4, 8, 11, 15, 18 and 22 of the 28-daytreatment cycle at a dose of about 10 mg, 20 mg, 40 mg, 80 mg, 160 mg,240 mg, 320 mg, or 400 mg.

In some embodiments, the treatment comprises administering theBcl-2/Bcl-xL inhibitor on days 1, 8, 15 and 22 of the 28-day treatmentcycle at a dose of about 10 mg, 20 mg, 40 mg, 80 mg, 160 mg, 240 mg, 320mg, or 400 mg.

In some embodiments, the the Bcl-2/Bcl-xL inhibitor is administeredthrough intravenous infusion.

In some embodiments, the Bcl-2/Bcl-xL inhibitor is administered at anamount from about 0.005 to about 500 mg/day, from about 0.05 to about250 mg/day or from about 0.5 to about 100 mg/day.

According to another aspect of the invention, provided herein is use ofa combination of a Bcl-2/Bcl-xL inhibitor and a chemotherapeutic agentfor the manufacture of a medicament for the treatment of cancer.

In some embodiments, the Bcl-2/Bcl-xL inhibitor is(R)-2-(1-(3-(4-(N-(4-(4-(3-(2-(4-chlorophenyl)-1-isopropyl-5-methyl-4-(methylsulfonyl)-1H-pyrrol-3-yl)-5-fluorophenyl)piperazin-1-yl)phenyl)sulfamoyl)-2-(trifluoromethylsulfonyl)phenylamino)-4-(phenylthio)butyl)piperidine-4-carbonyloxy)ethylphosphonicacid, or(R)-1-(3-(4-(N-(4-(4-(3-(2-(4-chlorophenyl)-1-isopropyl-5-methyl-4-(methylsulfonyl)-1H-pyrrol-3-yl)-5-fluorophenyl)piperazin-1-yl)phenyl)sulfamoyl)-2-(trifluoromethylsulfonyl)phenylamino)-4-(phenylthio)butyl)piperidine-4-carboxylicacid; and the chemotherapeutic agent is selected from the groupconsisting of docetaxel, 5-fluorouracil, panobinstat, paclitaxel,cisplatin, irinotecan, topotecan, and etoposide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the inhibiting effect of combination therapy of Compound 15(i.e., Compound 15 described in Table 1 below) and docetaxel on cellproliferation in breast cancer xenograft model of mice bearing humanMDA-MB-231 cell line.

FIG. 2 shows the anti-cancer effect of combination therapy of Compound15 and docetaxel in breast cancer xenograft model of mice bearing humanMDA-MB-231 cell line.

FIG. 3 shows the anti-cancer effect of combination therapy of Compound15 and docetaxel in small cell lung cancer xenograft model of micebearing human NCI-H146 cell line.

FIG. 4 shows the anti-cancer effect of combination therapy of Compound15 and docetaxel in prostate cancer xenograft model of mice bearinghuman PC3 cell line.

FIG. 5 shows the anti-cancer effect of combination therapy of Compound15 and docetaxel in gastric cancer xenograft model of mice bearing humanMGC80-3 cell line.

FIG. 6 shows the anti-cancer effect of combination therapy of Compound15 and docetaxel or cisplatin in gastric cancer xenograft model of micebearing human MGC80-3 cell line.

FIG. 7 shows the anti-cancer effect of combination therapy of Compound15 with irinotecan in colon cancer xenograft model of mice bearing humanSW620 cell line.

FIGS. 8(a) and 8(b) show the combination therapy of Compound 15 andpaclitaxel (FIG. 8(a)) or irinotecan (FIG. 8(b)) in a subcutaneousLU5250 SCLC PDX model.

FIGS. 9(a) and 9(b) show the combination therapy of Compound 15 andpaclitaxel (FIG. 9(a)) or irinotecan (FIG. 9(b)) in a subcutaneousLU5188 SCLC PDX model.

FIGS. 10(a) and 10(b) show the body weight change of the LU5250 modelmice after administration of the following drug combinations: Compound15+paclitaxel (FIG. 10(a)), Compound 15+irinotecan (FIG. 10(b)).

FIGS. 11(a) and 11(b) show the body weight change of the LUS188 modelmice after administration of the following drug combinations: Compound15+paclitaxel (FIG. 11(a)), Compound 15+irinotecan (FIG. 11(b)).

FIGS. 12(a), 12(b) and 12(c) show the combination therapy of Compound 15and paclitaxel (FIG. 12(a)), Compound 15 and irinotecan (FIG. 12(b)) orCompound 15 and cisplatin (FIG. 12(c)) in a subcutaneous human NCI-H146small cell lung cancer model.

FIGS. 13(a), 13(b) and 13(c) show the mean body weight change of micemodels obtained by inoculation of subcutaneous human NCI-H146 small celllung cancer cells, in response to administration of the followingcombination therapies: Compound 15+paclitaxel (FIG. 13(a)), Compound15+irinotecan (FIG. 13(b)) and Compound 15+cisplatin (FIG. 13(c)).

FIGS. 14(a), 14(b) and 14(c) show the anti-proliferative effect ofCompound 31 in combination with paclitaxel in human small cell lungcancer cell line NCI-H146 (FIG. 14(a)), NCI-H69 (FIG. 14(b)), NCI-H446(FIG. 14(c)).

FIGS. 15(a), 15(b) and 15(c) show the anti-proliferative effect ofCompound 31 in combination with topotecan in human small cell lungcancer cell line NCI-H146 (FIG. 15(a)), NCI-H69 (FIG. 15(b)), NCI-H446(FIG. 15(c)).

FIGS. 16(a) and 16(b) show the anti-proliferative effect of Compound 31in combination with cisplatin in human small cell lung cancer cell lineNCI-H146 (FIG. 16(a)), NCI-H69 (FIG. 16(b)).

FIGS. 17(a) and 17(b) show the anti-proliferative effect of Compound 31in combination with irinotecan in human small cell lung cancer cell lineNCI-H146 (FIG. 17(a)), NCI-H69 (FIG. 17(b)).

FIGS. 18(a) and 18(b) show the anti-proliferative effect of Compound 31in combination with etoposide in human small cell lung cancer cell lineNCI-H146 (FIG. 18(a)), NCI-H69 (FIG. 18(b)).

FIG. 19 shows that caspase 3/7 activity increases in the human smallcell lung cancer cell line NCI-H146 treated with Compound 31 incombination with paclitaxel.

FIG. 20 shows that caspase 3/7 activity increases in the human smallcell lung cancer cell line NCI-H446 treated with Compound 31 incombination with topotecan.

FIGS. 21(a) and 21(b) show that caspase 3/7 activity increases in thehuman small cell lung cancer cell lines NCI-H146 (FIG. 21(a)) andNCI-H69 (FIG. 21(b)) treated with Compound 31 in combination withetoposide.

FIGS. 22(a) and 22(b) show that caspase 3/7 activity increases in thehuman small cell lung cancer cell lines NCI-H146 (FIG. 22(a)) andNCI-H69 (FIG. 22(b)) treated with Compound 31 in combination withirinotecan.

FIGS. 23(a) and 23(b) show that caspase 3/7 activity increases in thehuman small cell lung cancer cell lines NCI-H146 (FIG. 23(a)) andNCI-H69 (FIG. 23(b)) treated with Compound 31 in combination withcisplatin.

FIGS. 24(a) and 24(b) show the combination therapy of Compound 15 andcisplatin (FIG. 24(a)) or docetaxel (FIG. 24(b)) in a subcutaneousnon-small cell lung cancer xenograft model of mice bearing humanNCI-H1975-L858R-T790M-C797S cell line.

FIG. 25 shows basal expression levels of Bcl-2 family proteins in AGSand NCI-N87 gastric cancer tumor cell lines.

FIGS. 26(a) and 26(b) show the inhibiting effect of Compound 31 on cellproliferation in both AGS (FIG. 26(a)) and NCI-N87 (FIG. 26(b)) celllines.

FIG. 27 shows that Compound 31 had no significant effect on cell cycle.

FIGS. 28(a) through 28(f) show the effects of Compound 15 at differentconcentrations on mouse xenograft tumor volume (FIG. 28(a)), tumorweight (FIG. 28(b)), tumor size (FIG. 28(c)), mouse body weight (FIG.28(d)), apoptosis-related proteins in mouse tumor tissues (FIG. 28(e))and apoptosis-related immunohistochemistry in mouse tumor tissues (FIG.28(f)).

FIG. 29(a) shows that Compound 31 in combination with 5-fluorouracil(5-FU) induced apoptosis in gastric cancer cells; FIG. 29(b) shows theeffect of combination therapy of Compound 31 and 5-FU onapoptosis-related proteins in gastric cancer cells.

FIGS. 30(a) through 30(f) show the effects of combination therapy ofCompound 15 and 5-fluorouracil (5-FU) on mouse xenograft tumor volume(FIG. 30(a)), tumor weight (FIG. 30(b)), tumor size (FIG. 30(c)), bodyweight (FIG. 30(d)), TUNEL detection in mouse tumor tissues (FIG. 30(e))and apoptosis-related immunohistochemistry in mouse tumor tissues (FIG.30(f)).

FIG. 31 shows the inhibiting effect of combination therapy of Compound31 and docetaxel (TXT) on cell proliferation in gastric cancer xenograftmodel of mice bearing NCI-N87 cell line.

FIG. 32 shows the inhibiting effect of combination therapy of Compound31 and panobinostat on cell proliferation in gastric cancer xenograftmodel of mice bearing HGC-27 cell line.

FIG. 33(a) shows the platelet (PTL) counts of the patients with thetreatment of Compound 15 at a dose of 400 mg (BIW), 320 mg (BIW) or 320mg (QW).

FIG. 33(b) shows the target lesion in liver of a partial response (PR)patient (patient #002-001) with SCLC at baseline, cycle 2 and cycle 10of the treatment with Compound 15.

FIG. 33(c) shows the duration of response for patients with PR or stabledisease (SD) after the treatment with Compound 15.

FIG. 33(d) shows mean pharmacokinetic (PK) profile of Compound 15 atdifferent dose levels, and the mean PK values of Compound 15 at a doseof 240 mg (QW).

DETAILED DESCRIPTION OF THE INVENTION

The invention will now be described in connection with specificembodiments. It should be understood that the present disclosure is notlimited to the following embodiments. Any changes or modifications maybe made by those skilled in the art without departing from the scope ofthe invention, and such modifications or improvements are also includedin the scope of the invention.

Pharmaceutical Composition

According to a first aspect of the present disclosure, provided hereinis a pharmaceutical composition comprising a Bcl-2/Bcl-xL inhibitor anda chemotherapeutic agent, and a pharmaceutically acceptable carrier. TheBcl-2/Bcl-xL inhibitor is a compound of the following formula (I), (II),(III) or (IV), or a pharmaceutically acceptable salt thereof:

wherein the A ring is

X, substituted or unsubstituted, is selected from the group consistingof alkylene, alkenylene, cycloalkylene, cycloalkenylene, andheterocycloalkylene;

Y is selected from the group consisting of (CH₂)_(n)—N(R^(a))₂ and

Q is selected from the group consisting of O, O(CH₂)₁₋₃, NR^(c),NR^(c)(C₁₋₃alkylene), OC(═O)(C₁₋₃alkylene), C(═O)O,C(═O)O(C₁₋₃alkylene), NHC(═O)(C₁₋₃alkylene), C(═O)NH, andC(═O)NH(C₁₋₃alkylene);

Z is O or NR^(c).

R₁ and R₂, independently, are selected from the group consisting of H,CN, NO₂, halo, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl,heteroaryl, heterocycloalkyl, OR′, SR′, NR′R″, COR′, CO₂R′, OCOR′,CONR′R″, CONR′SO₂R″, NR′COR″, NR′CONR″R′″, NR′C═SNR″R′″, NR′SO₂R″,SO₂R′, and SO₂NR′R″;

R₃ is selected from a group consisting of H, alkyl, cycloalkyl, alkenyl,cycloalkenyl, alkynyl, aryl, heteroaryl, heterocycloalkyl, OR′, NR′R″,OCOR′, CO₂R′, COR′, CONR′R″, CONR′SO₂R″, C₁₋₃alkyleneCH(OH)CH₂OH, SO₂R′,and SO₂NR′R″;

R′, R″, and R′″, independently, are H, alkyl, cycloalkyl, alkenyl,cycloalkenyl, alkynyl, aryl, heteroaryl, C₁₋₃alkyleneheterocycloalkyl,or heterocycloalkyl;

R′ and R″, or R″ and R′″, can be taken together with the atom to whichthey are bound to form a 3 to 7 membered ring;

R₄ is hydrogen, halo, C₁₋₃alkyl, CF₃, or CN;

R₅ is hydrogen, halo, C₁₋₃alkyl, substituted C₁₋₃alkyl, hydroxyalkyl,alkoxy, or substituted alkoxy;

R₆ is selected from the group consisting of H, CN, NO₂, halo, alkyl,cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl,heterocycloalkyl, OR′, SR′, NR′R″, CO₂R′, OCOR′, CONR′R″, CONR′SO₂R″,NR′COR″, NR′CONR″R′″, NR′C═SNR″R′″, NR′SO₂R″, SO₂R′, and SO₂NR′R″;

R₇, substituted or unsubstituted, is selected form the group consistingof hydrogen, alkyl, alkenyl, (CH₂)₀₋₃cycloalkyl, (CH₂)₀₋₃cycloalkenyl,(CH₂)₀₋₃heterocycloalkyl, (CH₂)₀₋₃aryl, and (CH₂)₀₋₃heteroaryl;

R₈ is selected form the group consisting of hydrogen, halo, NO₂, CN,CF₃SO₂, and CF₃;

R_(a) is selected from the group consisting of hydrogen, alkyl,heteroalkyl, alkenyl, hydroxyalkyl, alkoxy, substituted alkoxy,cycloalkyl, cycloalkenyl, and heterocycloalkyl;

R_(b) is hydrogen or alkyl;

R_(c) is selected from the group consisting of hydrogen, alkyl,substituted alkyl, hydroxyalkyl, alkoxy, and substituted alkoxy;

n, r, and s, independently, are 1, 2, 3, 4, 5, or 6;

R₂₁ is SO₂R₂′,

R₂₂ is alkyl, preferably C₁₋₄ alkyl, more preferably methyl, propyl, orisopropyl,

R₂₃ is alkyl, preferably C₁₋₄ alkyl, more preferably methyl, propyl, orisopropyl,

R₂₄ is halogen, preferably fluoride, chloride,

R₂₅ is halogen, preferably fluoride, chloride,

R₂₆ is selected from H, halogen, alkyl, preferably fluoride, chloride,C₁₋₄ alkyl, more preferably methyl, propyl, isopropyl

R_(21b) is H or alkyl, preferably C₁₋₄alkyl, more preferably methyl,propyl, or isopropyl,

n₂, r₂ and s₂ are independently 1, 2, 3, 4, 5 or 6, more preferably, r₂and s₂ are both 2 and n₂ is 3, 4 or 5, more preferably, all of n₂, r₂and s₂ are 2; and

R₂′ is alkyl, preferably C₁₋₄ alkyl, more preferably methyl, propyl, orisopropyl.

Bcl-2/Bcl-xL Inhibitor

As used herein, the term “Bcl-2/Bcl-xL” means Bcl-2, Bcl-xL, or Bcl-2and Bcl-xL, i.e., Bcl-2 and/or Bcl-xL.

As used herein, the term “alkyl” refers to straight chain and branchedsaturated C₁₋₁₀ hydrocarbon groups, preferably C₁₋₆ hydrocarbon groups,non-limiting examples of which include methyl, ethyl, and straight chainand branched propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, anddecyl groups. The term C_(n) means the alkyl group has “n” carbon atoms.The term C_(n-p) means that the alkyl group contains “n” to “p” carbonatoms.

The term “alkylene” refers to an bivalent alkyl group having a generalformula of —(CH₂)_(n)—, wherein n is an integer selected from 1-10. Analkyl, e.g., methyl, or alkylene, e.g., —CH₂— group can be unsubstitutedor substituted with halo, trifluoromethyl, trifluoromethoxy, hydroxy,alkoxy, nitro, cyano, alkylamino, or amino groups, for example.

The term “alkenyl” is defined identically as “alkyl,” except forcontaining a carbon-carbon double bond, e.g., ethenyl, propenyl, andbutenyl. The term “alkenylene” is defined identically to “alkylene”except for containing a carbon-carbon double bond. The term “alkynyl”and “alkynylene” are defined identically as “alkyl” and “alkylene”except the group contains a carbon-carbon triple bond.

As used herein, the term “halo” is defined as fluoro, chloro, bromo, andiodo.

The term “hydroxy” is defined as —OH.

The term “alkoxy” is defined as —OR, wherein R is alkyl.

The term “amino” is defined as —NH₂, and the term “alkylamino” isdefined as —NR₂, wherein at least one R is alkyl and the second R isalkyl or hydrogen.

The term “nitro” is defined as —NO₂.

The term “cyano” is defined as —CN.

The term “trifluoromethyl” is defined as —CF₃.

The term “trifluoromethoxy” is defined as —OCF₃.

As used herein, groups such as

is an abbreviation for

As used herein, the term “aryl” refers to a monocyclic or polycyclicaromatic group, preferably a monocyclic or bicyclic aromatic group,e.g., phenyl or naphthyl. Unless otherwise indicated, an aryl group canbe unsubstituted or substituted with one or more, and in particular oneto four, groups independently selected from, for example, halo, alkyl,alkenyl, —OCF₃, —CF₃, —NO₂, —CN, —NC, —OH, alkoxy, amino, alkylamino,—CO₂H, —CO₂alkyl, —OCOalkyl, aryl, and heteroaryl.

As used herein, the term “heteroaryl” refers to a monocyclic or bicyclicring system containing one or two aromatic rings and containing at leastone nitrogen, oxygen, or sulfur atom in an aromatic ring. Unlessotherwise indicated, a heteroaryl group can be unsubstituted orsubstituted with one or more, and in particular one to four,substituents selected from, for example, halo, alkyl, alkenyl, —OCF₃,—CF₃, —NO₂, —CN, —NC, —OH, alkoxy, amino, alkylamino, —CO₂H, —CO₂alkyl,—OCOalkyl, aryl, and heteroaryl.

As used herein, the term “cycloalkyl” means a monocyclic aliphatic ringcontaining 3-8 carbon atoms. The term “heterocycloalkyl” means amonocyclic or bicyclic ring system containing at least one nitrogen,oxygen, or sulfur atom in the ring system. The terms “heteroaryl” and“heterocycloalkyl” encompass ring systems containing at least one oxygenatom, nitrogen atom, or sulfur atom, and includes ring systemscontaining oxygen and nitrogen atoms, oxygen and sulfur atoms, nitrogenand sulfur atoms, and nitrogen, oxygen, and sulfur atoms.

In the above formula (I), (II) or (III), in some embodiments, R₁ and R₂or R₂ and R₃ may together form a ring. In other embodiments, R′ and R″or R″ and R″″ may form a 3-7 membered ring with the atoms to which theyare attached.

In some preferred embodiments, X is alkylene, and in preferredembodiments, is C₁₋₃ alkylene.

In some embodiments, Y is

In preferred embodiments, n is 2. In other preferred embodiments, R_(b)is hydrogen or C₁₋₃ alkyl.

In still other preferred embodiments, Q is O, O(CH₂)₁₋₃, C(═O)O(CH₂)₁₋₃,OC(═O)(CH₂)₁₋₃, or C(═O)O(C₃H₇)₁₋₃. In some embodiments, Q is O, OCH₂,C(═O)OCH₂, C(═O)O(CH₂)₂, C(═O)O(CH₂)₃, OC(═O)CH₂ or C(═O)O(CH(CH₃)CH₂).

In some embodiments, Z is O, NH, or N(C₁₋₃alkyl). In preferredembodiments, Z is O, NH, or NCH₃.

In some embodiments, R₁ is SO₂R′, SO₂NR′R″, NR′SOR″, H, or alkyl. Insome preferred embodiments, R₁ is SO₂(C₁₋₃alkyl), SO₂N(C₁₋₃alkyl)₂,NHSO₂(C₁₋₃alkyl), H, or C₁₋₃alkyl. One preferred embodiment of R₁ isSO₂CH₃.

In some embodiments, R₂ and R₃, independently, are H, C₁₋₃alkyl, orcycloalkyl. R₂ also can be halo. In some preferred embodiments, R₂ andR₃, independently, are methyl, ethyl, n-propyl, isopropyl, cyclopentyl,or cyclohexyl. R₂ also can be Cl or F.

In some embodiments, R₄ is H or halo, preferably H, Cl, or F. In otherembodiments, R₅ is H, halo or C₁₋₃alkyl, preferrably H, methyl, ethyl,n-propyl, isopropyl, F, or Cl. In other embodiments, R₆ is H, halo,alkyl, or cycloalkyl. In some preferred embodiments, R₆ is H, F, Cl,C₁₋₃alkyl, cyclopentyl, or cyclohexyl.

In some embodiments, R₇ is (CH₂)₀₋₃cycloalkyl or(CH₂)₀₋₃heterocycloalkyl. In a preferred embodiment, R₇ is(CH₂)₀₋₃cycloalkyl, optionally substituted with —OH. In one embodiment,R₇ is

In some embodiments, R₈ is CF₃SO₂ or CF₃. In various embodiments, R_(a),R_(b), and R_(c), independently, are H or C₁₋₃alkyl.

Additionally, salts, hydrates, solvates and active metabolites of thepresent compounds of formula (I), (II), (III) or (IV) also are includedin the present disclosure and can be used in the methods disclosedherein. The present disclosure further includes all possiblestereoisomers and geometric isomers of the compounds of structuralformula (I), (II), (III) or (IV). The present disclosure includes bothracemic compounds and optically active isomers. When a compound ofstructural formula (I), (II), (III) or (IV) is desired as a singleenantiomer, it can be obtained either by resolution of the final productor by stereospecific synthesis from either isomerically pure startingmaterial or use of a chiral auxiliary reagent, for example, see Z. Ma etal., Tetrahedron: Asymmetry, 8(6), pages 883-888 (1997). Resolution ofthe final product, an intermediate, or a starting material can beachieved by any suitable method known in the art. Additionally, insituations where tautomers of the compounds of structural formula (I),(II), (III) or (IV) are possible, the present disclosure is intended toinclude all tautomeric forms of the compounds.

As used herein, the term “pharmaceutically acceptable salts” refers tosalts or zwitterionic forms of the compounds of structural formula (I),(II), (III) or (IV). Salts of compounds of formula (I), (II), (III) or(IV) can be prepared during the final isolation and purification of thecompounds or separately by reacting the compound with an acid having asuitable cation. The pharmaceutically acceptable salts of compounds ofstructural formula (I), (II), (III) or (IV) can be acid addition saltsformed with pharmaceutically acceptable acids. Examples of acids whichcan be employed to form pharmaceutically acceptable salts includeinorganic acids such as nitric, boric, hydrochloric, hydrobromic,sulfuric, and phosphoric, and organic acids such as oxalic, maleic,succinic, and citric. Nonlimiting examples of salts of compounds of theinvention include, but are not limited to, the hydrochloride,hydrobromide, hydroiodide, sulfate, bisulfate, 2-hydroxyethansulfonate,phosphate, hydrogen phosphate, acetate, adipate, alginate, aspartate,benzoate, bisulfate, butyrate, camphorate, camphorsulfonate,digluconate, glycerolphsphate, hemisulfate, heptanoate, hexanoate,formate, succinate, fumarate, maleate, ascorbate, isethionate,salicylate, methanesulfonate, mesitylenesulfonate, naphthylenesulfonate,nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate,persulfate, 3-phenylproprionate, picrate, pivalate, propionate,trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate,paratoluenesulfonate, undecanoate, lactate, citrate, tartrate,gluconate, methanesulfonate, ethanedisulfonate, benzene sulphonate, andp-toluenesulfonate salts. In addition, available amino groups present inthe compounds of the invention can be quaternized with methyl, ethyl,propyl, and butyl chlorides, bromides, and iodides; dimethyl, diethyl,dibutyl, and diamyl sulfates; decyl, lauryl, myristyl, and stearylchlorides, bromides, and iodides; and benzyl and phenethyl bromides.

In accordance with the foregoing, any reference to a compound of theinvention presented herein is intended to include a compound of formula(I), (II), (III) or (IV), and pharmaceutically acceptable salts,hydrates, solvates or active metabolite thereof. The term “activemetabolite” as used herein refers to a metabolite of a compound offormula (I), (II), (III) or (IV) in the human body.

Specific compounds of the present disclosure include, but are notlimited to, compounds having the structure formulae set forth below inTable 1.

TABLE 1 Compound # Formula 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

The Bcl-2/Bcl-xL inhibitor of the present disclosure is preferably(R)-2-(1-(3-(4-(N-(4-(4-(3-(2-(4-chlorophenyl)-1-isopropyl-5-methyl-4-(methylsulfonyl)-1H-pyrrol-3-yl)-5-fluorophenyl)piperazin-1-yl)phenyl)sulfamoyl)-2-(trifluoromethylsulfonyl)phenylamino)-4-(phenylthio)butyl)piperidine-4-carbonyloxy)ethylphosphonicacid (i.e., compound 15, in the above table, sometimes abbreviated as“compound 15”) or a pharmaceutically acceptable salt thereof, asrepresented by the following structural formula:

Compound 15 selectively binds to Bcl-2, Bcl-xl, and Bcl-w proteins withhigh affinity, and has an IC50 of 1.6 nM, 4.4 nM, and 9.3 nM,respectively. Compound 15 binds weakly to Mcl-1. Having chemicalstructural modification, Compound 15 effectively reduces the platelettoxicity defects of the first-generation BCL-2 inhibitors in the bloodcirculation, and is capable of inducing specific enzyme activation intissues to effectively kill tumor cells. Its platelet toxicity isreduced by 10-30 times, but the activity is about 10 times that of thefirst generation BCL-2 inhibitor. Compound 31 is an active metabolite ofCompound 15. Compound 15 is a novel second generation protein inhibitortargeting BCL-2.

The Bcl-2/Bcl-xL inhibitor of the present disclosure may also preferablybe(R)-1-(3-(4-(N-(4-(4-(3-(2-(4-chlorophenyl)-1-isopropyl-5-methyl-4-(methylsulfonyl)-1H-pyrrol-3-yl)-5-fluorophenyl)piperazin-1-yl)phenyl)sulfamoyl)-2-(trifluoromethylsulfonyl)phenylamino)-4-(phenylthio)butyl)piperidine-4-carboxylicacid (i.e., compound 31 in the above table, sometimes abbreviated as“compound 31”) or a pharmaceutically acceptable salt thereof, asrepresented by the following structural formula:

The above Bcl-2/Bcl-xL inhibitor of the pharmaceutical composition ofthe present disclosure can be synthesized according to the methoddescribed in WO2014/113413A1.

Chemotherapeutic Agent

The term “chemotherapeutic drug” is a biological (macromolecule) orchemical (small molecule) compound that can be used to treat cancer. Thetypes of chemotherapeutic drugs include, but are not limited to, histonedeacetylase inhibitor (HDACI), alkylating agents, antimetabolites,alkaloids, cytotoxic/anti-cancer antibiotics, topoisomerase inhibitors,tubulin inhibitors, proteins, antibodies, kinase inhibitors, and thelike. Chemotherapeutic drugs include compounds for targeted therapy andnon-targeted compounds of conventional chemotherapy.

Non-limiting examples of chemotherapeutic drugs include: erlotinib,afatinib, docetaxel, adriamycin, 5-FU (5-fluorouracil), panobinostat,gemcitabine, cisplatin, carboplatin, paclitaxel, bevacizumab,trastuzumab, pertuzumab, metformin, temozolomide, tamoxifen,doxorubicin, rapamycin, lapatinib, hydroxycamptothecin, trimetinib.Further examples of chemotherapeutic drugs include: oxaliplatin,bortezomib, sunitinib, letrozole, imatinib, PI3K inhibitor, fulvestrant,leucovorin, lonafarnib, sorafenib, gefitinib, crizotinib, irinotecan,topotecan, valrubicin, vemurafenib, telbivinib, capecitabine,vandetanib, chloranmbucil, panitumumab, cetuximab, rituximab,tositumomab, temsirolimus, everolimus, pazopanib, canfosfamide,thiotepa, cyclophosphamide; alkyl sulfonates e.g., busulfan, improsulfanand piposulfan; ethyleneimine, benzodopa, carboquone, meturedopa,uredopa, methylmelamine, including altretamine, triethylenemelamine,triethyl phosphamide, triethyl thiophosphamide and trimethylenemelamine;bullatacin, bullatacinone; bryostatin; callystatin, CC-1065 (includingits adozelesin, carzelesin, bizelesin synthetic analogue), cryptophycin(in particular, cryptophycin 1 and cryptophycin 8); dolastatin,duocarmycin (including synthetic analogue KW-2189 and CB1-TM1);eleutherobin; pancratistatin, sarcodictyin, spongistatin; nitrogenmustards, e.g., chlorambucil, chlornaphazine, cyclophosphamide,estramustine, ifosfamide, bis-chloroethyl-methylamine,Mechlorethaminoxide (melphalan, novembichin, phenesterine,prednimustine, trofosfamide, uramustine, nitrosourea, e.g., carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimnustine,antibiotics, e.g., enediyne antibiotics (e.g., calicheamicin,calicheamicin γ1I, calicheamicin ωI1, dynemicin, dynemicin A;diphosphate, e.g, clodronate, esperamicin, and neocarzinostatinchromophore and related chromoprotein enediyne antibiotics chromophore),aclacinomycin, actinomycin, all-trans retinoic acid, anthramycin,azaserine, bleomycin, actinomycin C, carabicin, carminomycin,carzinophilin, chromomycinis, actinomycin D, daunorubicin,deoxy-fluorouridine, detorubicin, 6-dizao-5-oxo-L-norleucine,morpholino-doxorubicin, cyno-morpholino-doxorubicin,2-pyrroline-doxorubicin, eoxy doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin,olivomycin, peplomycin, porfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; antimetabolite, e.g., methotrexate; folateanalogue, e.g., dimethylfolate, methotrexate, pteropterin, trimetrexate,purine analogue, e.g., fludarabine, 6-mercaptopurine, methotrexate,thiamiprine, tioguanine; pyrimidine analogue, e.g., ancitabine,azacitidine, azathioprine, bleomycin, 6-nitrouridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgen, calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; antiadrenergic agent, e.g.aminoglutethimide, mitotane, trilostane; folate supplement, e.g.folinate; aceglatone; aldophosphamide glycoside; aminolevulinic acid;eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine,demecolcine, diaziquone, eflornithine, elliptinium acetate, epothilone,etoglucid; gallium nitrate; hydroxycarbamide; lentinan, lonidainine,maytansinoid, maytansine, ansamitocin, mitoguazone, mitoxantrone,mopidamol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone,podophyllinic acid; 2-ethylhydrazine; procarbazine, PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.), razoxane, rhizoxin,sizofiran, spirogermanium, tenuazonic acid, triaziquone;2,2′,2″-trichloro-triethylamine; trichothecene (in particular, T-2toxin,verracurin A, roridin A and anguidine); urethane, vindesine,dacarbazine, mannomustine; dibromomannitol; dibromodulcitol; pipobroman,gacytosine, arabinoside (“Ara-C”); cyclophosphamide; thiotepa;tioguanine; 6-mercaptopurine; methotrexate; Vinblastine; etoposide,ifosfamide, mitoxantrone, vincristine, vinorelbine, novantrone;emetrexed; teniposide, edatrexate, daunomycin; aminopterin; ibandronate;CPT-11; topoisomerase inhibitor RFS 2000; DMFO, retinoid, e.g., Retinoicacid; and a pharmaceutically acceptable salt or derivative thereof.

The chemotherapeutic drug used herein is preferably actinomycin,all-trans retinoic acid, azacitidine, azathioprine, bleomycin,bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil,cyclophosphamide, cytosine arabinoside, daunorubicin, docetaxel,deoxyfluorouridine, doxorubicin, epirubicin, epothilone, etoposide,fluorouracil (e.g. 5-fluorouracil), panobinstat, gemcitabine,hydroxyurea, idarubicin, imatinib, irinotecan, nitrogen mustard,Mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel,pemetrexed, teniposide, thioguanine, topotecan, valrubicin, vemurafenib,vinblastine, vincristine, vindesine, vinorelbine or hydroxycamptothecin,more preferably docetaxel, cisplatin, irinotecan, doxorubicin,docetaxel, cisplatin, gemcitabine, irinotecan or hydroxycamptothecin,even more preferably docetaxel, fluorouracil (e.g. 5-fluorouracil),panobinstat, cisplatin, irinotecan, paclitaxel, topotecan, or etoposide,most preferably is docetaxel, fluorouracil (e.g. 5-fluorouracil), orpanobinstat.

Treatment of Cancer

The pharmaceutical composition described herein can be used for thetreatment of cancer, which include but not limited to adrenal cancer,lymphoepithelial neoplasia, adenoid cell carcinoma, lymphoma, acousticneuroma, acute lymphocytic leukemia, acral lentiginous melanoma, acutemyeloid leukemia, acrospiroma, chronic lymphocytic leukemia, acuteeosinophilic leukemia, liver cancer, acute erythrocyte leukemia, smallcell lung cancer, acute lymphocytic leukemia, non-small cell lungcancer, acute megakaryoblastic leukemia, MALT lymphoma, acute monocyticleukemia, malignant fibrous histiocytoma, acute promyelocytic leukemia,malignant peripheral schwannomas, mantle cell lymphoma, adenocarcinoma,marginal zone B-cell lymphoma, malignant hippocampal tumor, adenoidcystic carcinoma, gland tumor, adenoma-like odontogenic tumor, mast cellleukemia, adenosquamous carcinoma, mediastinal germ cell tumor, adiposetissue tumor, breast medullary carcinoma, adrenocortical carcinoma,medullary thyroid carcinoma, adult T cell leukemia/lymphoma,Medulloblastoma, invasive NK cell leukemia, melanoma, AIDS-relatedlymphoma, meningioma, lung rhabdomyosarcoma, Merkel cell carcinoma,alveolar soft tissue sarcoma, mesothelioma, ameloblastoma, metastaticurothelial carcinoma, anaplastic large cell lymphoma, mixed Mülleriantumor, thyroid undifferentiated carcinoma, mucinous neoplasm,angioimmunoblastic T-cell lymphoma, multiple myeloma, angiomyolipoma,muscle tissue tumor, angiosarcoma, mycosis fungoides, astrocytoma,myxoid liposarcoma, atypical deformed rhabdoid tumor, myxoma, B-cellchronic lymphocytic leukemia, mucinous sarcoma, B-cell lymphoblasticleukemia, nasopharyngeal carcinoma, B-cell lymphoma, schwannomas, basalcell carcinoma, neuroblastoma, biliary tract cancer, neurofibromatosis,bladder cancer, neuroma, blastoma, nodular melanoma, bone cancer, eyecancer, Brenner tumor, oligodendroxoma, brown tumor, oligodendroglioma,Burkitt's lymphoma, eosinophilic breast cancer, brain cancer, opticnerve tumor cancer, oral cancer carcinoma in situ, osteosarcoma,carcinosarcoma, ovarian cancer, cartilage tumor, pulmonary sulcus tumor,papillary thyroid carcinoma, myeloma, paraganglioma, chondroma, pinealblastoma, chordoma, pineal cell tumor, choriocarcinoma, pituitary tumor,choroid plexus papilloma, pituitary adenoma, kidney clear cell sarcoma,pituitary tumor, craniopharyngioma, plasmacytoma, cutaneous T-celllymphoma, multiple embryonic cell tumor, cervical cancer, precursor Tlymphoblastic lymphoma, colorectal cancer, primary central nervoussystem lymphoma, Degos disease, primary effusion lymphoma, proliferativesmall round cell tumor, primary preformed peritoneal cancer, diffuselarge B-cell lymphoma, prostate cancer, embryonic dysplasia ofneuroepithelial neoplasia, pancreatic cancer, anaplastic cell tumor,pharyngeal carcinoma, embryonic carcinoma, peritoneal pseudomyxoma,endocrine gland tumor, renal cell carcinoma, enteropathy-associatedT-cell lymphoma, endodermal sinus tumor, renal medullary carcinoma,retinoblastoma, esophageal cancer, rhabdomyosarcoma, endadelphos,rhabdomyosarcoma, fibroids, Richter's transformation, fibrosarcoma,rectal cancer, follicular lymphoma, sarcoma, follicular thyroid cancer,schwannoma, ganglion cell tumor, seminoma, gastrointestinal cancer,Sertoli cell tum, germ cell tumor, sex cord-gonadal stromal tumor,pregnancy choriocarcinoma, signet ring cell carcinoma, giant cellfibroblastoma, skin cancer, bone giant cell tumor of bone, small blueround cell tumor, glioma, small cell carcinoma, glioblastoma multiforme,soft tissue sarcoma, glioma, somatostatin tumor, glioma brain, sootwart, pancreatic high glucagonoma, spinal tumor, Gonadoblastoma, spleenmarginal lymphoma, granulosa cell tumor, squamous cell carcinoma,estrogen tumor, synovial sarcoma, gallbladder cancer, Sezary disease,gastric cancer, small intestine cancer, hairy cell leukemia, squamouscell carcinoma, hemangioblastoma, gastric cancer, head and neck cancer,T-cell lymphoma, vascular epithelioma, testicular cancer, hematologicalmalignancies, sarcoma, hepatoblastoma, thyroid cancer, hepatosplenicT-cell lymphoma, transitional cell carcinoma, Hodgkin's lymphoma,laryngeal cancer, non-Hodgkin's lymphoma, urachal cancer, invasivelobular carcinoma, genitourinary cancer, intestinal cancer, urothelialcarcinoma, renal cancer, uveal melanoma, laryngeal cancer, uterinecancer, malignant freckle-like sputum, verrucous carcinoma, lethalmidline granuloma, visual pathway glioma, leukemia, vulvar cancer,testicular stromal tumor, vaginal cancer, liposarcoma, Waldenstrom'smacroglobulinemia Disease, lung cancer, adenolymphoma lymphangioma,nephroblastoma, lymphangisarcoma.

The above cancer is preferably lung cancer (for example, small cell lungcancer, non-small cell lung cancer), breast cancer, glioma, gastriccancer, prostate cancer, pancreatic cancer, liver cancer, colon cancer,acute lymphocytic leukemia, more preferably breast cancer, lung cancer(for example, small cell lung cancer, non-small cell lung cancer),prostate cancer, gastric cancer, colon cancer. In certain embodiments,the breast cancer is a triple negative breast cancer. Triple negativebreast cancer refers to breast cancer where expression of estrogenreceptor, progesterone receptor, and HER2 receptor are not detectable inthe cancer cells.

Other diseases and conditions (including cancer) that can be treated byadministering the pharmaceutical compositions of the present disclosureare disclosed in U.S. Patent Publication No. 2007/0027135, U.S. Pat. No.7,432,304, U.S. Patent Publication No. 2010/0278921, WO 2012/017251A1,and WO 2014/113413A1. They are incorporated herein in their entirety.

In certain embodiments, the cancer is a metastatic solid tumor.

In certain embodiments, the cancer is Bcl-2/Bcl-xL and Bax positive.“Bcl-2/Bcl-xL and Bax positive” means that it is characterized byBcl-2/Bcl-xL and Bax gene amplification or Bcl-2/Bcl-xL and Bax proteinoverexpression, and therefore the cancerous or malignant cells ortissues have abnormally high levels of Bcl-2/Bcl-xL and Bax genes and/orBcl-2/Bcl-xL and Bax proteins than normal healthy cells. In certainembodiments, one or more genetic mutations can occur in multiple typesof cancer, resulting in cancer cells producing excessive Bcl-2/Bcl-xLand Bax proteins.

In certain embodiments, the patient's cancer is diagnosed asBcl-2/Bcl-xL and Bax positive. The patient can be diagnosed by methodswell known in the art, such as by methods of hybridization detectionusing nucleic acid probes, methods for nucleic acid amplification,immunoassay methods for protein level, and the like. In certainembodiments, the cancer is gastric cancer. In certain embodiments, thegastric cancer is Bcl-2/Bcl-xL and Bax positive gastric cancer.Bcl-2/Bcl-xL and Bax-positive gastric cancer are characterized in havinggastric cancer cells having Bcl-2/Bcl-xL and Bax gene amplification orBcl-2/Bcl-xL and Bax protein overexpression. In a preferred embodiment,the gastric cancer patient simultaneously overexpresses Bcl-2/Bcl-xL andBax, i.e., the patient has Bcl-2/Bcl-xL and Bax positive gastric cancer.

In certain embodiments, the cancer is resistant to an EGFR inhibitor.

“Epidermal growth factor receptor” or “EGFR” is a cell surface tyrosinekinase receptor. EGFR is a protein product of the growth-promotingoncogene erbB or ErbB1, a member of the ERBB family of theprotooncogenes family, which plays a key role in the development andprogression of many types of cancers in human. In particular, enhancedexpression of EGFR was observed in breast cancer, bladder cancer, lungcancer, head cancer, cervical cancer and gastric cancer, andglioblastoma. Clinically, ERBB oncogene amplification and/or receptoroverexpression in tumors has been reported to be associated with diseaserecurrence and poor patient prognosis, as well as with response totherapies.

An EGFR inhibitor refers to a molecule capable of inhibiting tyrosinekinase activity of EGFR, and may be a small molecule compound or abiological macromolecule such as an antibody or an antibody fragment orthe like. Exemplary small molecule EGFR inhibitors include, but are notlimited to, first generation EGFR inhibitors such as gefitinib,erlotinib, ectinib, second generation EGFR inhibitors such as afatinib,dacomitib, imatinib and lapatinib, third-generation EGFR inhibitors suchas oxifitinib (AZD9291), nazatinib, rociletinib, naquotinib and thelike. Exemplary macromolecular EGFR inhibitors include, but are notlimited to, cetuximab and panitumumab.

In some embodiments, the EGFR inhibitor is selected from: gefitinib,erlotinib, ectinib, afatinib, dacomitinib, imatinib, lapatinib,osimertinib (AZD9291), nazatinib, rociletinib, naquotinib, vandetanib,neratinib, pelitinib, canertinib, briatininib, PKC412, Go6976,mavelertinib, olmutinib, WZ4002, TAS2913, cetuximab, panitumumab,avitinib, HS-10296 and TQB3804. In some embodiments, the EGFR inhibitoris osimertinib.

Drug resistance as used herein refers to being refractory ornon-responsive to a therapeutic agent, such as an EGFR inhibitor. Forexample, the number of tumor cells is increased despite of being treatedwith a therapeutic agent. In certain embodiments, the cancer resistantto EGFR inhibitor is lung cancer (e.g. small cell lung cancer ornon-small cell lung cancer).

In certain embodiments, the cancer (e.g. lung cancer) is characterizedin expressing a mutated EGFR The mutated EGFR has one or more mutationsselected from the group consisting of L858R, T790M, C797S, and EGFR geneexon 20 insertion. The standard amino acid sequence of EGFR can be foundin the sequence shown in accession number P00533-1 in the SwissProtdatabase, and the sequence shown in accession number NP_005219.2 in theNCBI database. The position of the mutation of EGFR in the presentapplication is based on the position in the standard amino acid sequenceof EGFR described above. For example, L858R, T790M and C797S refer toleucine at the position 858 of EGFR sequence replaced by arginine,threonine at the position 790 replaced by methionine, and cysteine atthe position 797 replaced by serine, respectively. The L858R mutation islocated in exon 21 of EGFR. The first generation of EGFR inhibitors cantreat patients with EGFR exon 19 deletion or exon 21 L858R mutation, butpatients are prone to develop drug resistance. The T790M mutation inEGFR exon 20 was found to be the most common mutation associated withthe development of the drug resistance, and T790M will in turn conferresistance to the inhibition by EGFR tyrosine kinase inhibitors.Third-generation EGFR inhibitors are effective on EGFR with a T790Mmutation. However, the development of the resistance to third-generationEGFR inhibitors has also been observed, including mutations in thecysteine residue at the position 797.

In certain embodiments, the patient is diagnosed as expressing a mutatedEGFR. The patient can be diagnosed by methods well known in the art, forexample, by hybridization-based methods using nucleic acid probes thatspecifically distinguish mutant EGFR and wild-type EGFR, by nucleic acidamplification-based methods, and by detection methods using antibodiesthat specifically distinguish between mutant EGFR and wild type EGFR,and the like.

It is surprisingly found in the present disclosure that the combinationof a Bcl-2/Bcl-xL inhibitor and a chemotherapeutic agent provided hereinis useful for the treatment of cancers having an EGFR mutation,including but not limited to cancers having the above EGFR mutation. Incertain embodiments, the Bcl-2/Bcl-xL inhibitor is Compound 15 orCompound 31. In certain embodiments, the chemotherapeutic agentcomprises a tubulin inhibitor. In certain embodiments, thechemotherapeutic agent is docetaxel, fluorouracil (e.g. 5-fluorouracil),panobinstat, or cisplatin.

In a preferred embodiment, the pharmaceutical composition of theinvention comprises a combination of Compound 15 or Compound 31 anddocetaxel. It is unexpectedly found by the inventors that the use of thecombination of Compound 15 or Compound 31 and docetaxel produces asignificant anti-cancer effect superior to Compound 15, Compound 31 orDocetaxel alone.

In a preferred embodiment, the pharmaceutical composition of theinvention comprises a combination of Compound 15 or Compound 31 and5-fluorouracil. It is unexpectedly found by the inventors that thecombination of Compound 15 or Compound 31 with 5-fluorouracil produces asignificantly superior anti-cancer effect superior to Compound 15,Compound 31 or 5-fluorouracil alone.

In a preferred embodiment, the pharmaceutical composition of theinvention comprises a combination of Compound 15 or Compound 31 and ahistone acetylation inhibitor (panobinstat). It is unexpectedly found bythe inventors that the use of the combination of Compound 15 or Compound31 and panobinstat produces a significantly superior anti-cancer effectsuperior to Compound 15, Compound 31 or pinobinstat alone.

In a preferred embodiment, the pharmaceutical composition of theinvention comprises a combination of Compound 15 or Compound 31 andcisplatin. It is unexpectedly found by the inventors that the use of thecombination of Compound 15 or Compound 31 and cisplatin produces asignificant anti-cancer effect superior to Compound 15, Compound 31 orcisplatin alone.

In a preferred embodiment, the pharmaceutical composition of theinvention comprises a combination of Compound 15 or Compound 31 andirinotecan. It is unexpectedly found by the inventors that the use ofthe combination of Compound 15 or Compound 31 and irinotecan produces asignificant anti-cancer effect superior to Compound 15, Compound 31 oririnotecan alone.

In a preferred embodiment, the pharmaceutical composition of theinvention comprises a combination of Compound 15 or Compound 31 andpaclitaxel. It is unexpectedly found by the inventors that the use ofthe combination of Compound 15 or Compound 31 and paclitaxel produces asignificant anti-cancer effect superior to Compound 15, Compound 31 orpaclitaxel alone.

In a preferred embodiment, the pharmaceutical composition of theinvention comprises a combination of Compound 15 or Compound 31 andtopotecan. It is unexpectedly found by the inventors that the use of thecombination of Compound 15 or Compound 31 and topotecan produces asignificant anti-cancer effect superior to Compound 15, Compound 31 ortopotecan alone.

Method of Treatment

According to a second aspect of the invention, provided herein is amethod for treating cancer comprising administering a therapeuticallyeffective amount of a Bcl-2/Bcl-xL inhibitor and a therapeuticallyeffective amount of a chemotherapeutic agent to an individual in needthereof, wherein the Bcl-2/Bcl-xL inhibitor and the chemotherapeuticagent are respectively as defined above. The cancer is also as definedabove.

The term “treatment” as used herein refers to eliminating, reducing orameliorating a disease or condition and/or a symptom associatedtherewith. For example, “treatment of cancer” includes treating,suppressing cancer, reducing its severity, reducing its risk, orinhibiting its metastasis. Although not excluded, treatment of a diseaseor condition does not require that the disease, condition, or symptomsassociated therewith be completely eliminated. The term “treatment” asused herein may include “prophylactic treatment”, which refers toreducing the possibility of recurrence of a disease or condition, orreducing the possibility of relapse of a previously controlled diseaseor condition, in a subject who is not afflicted with a disease but atrisk, or who is susceptible to recurrence of the disease or condition,or who is at risk or susceptible to relapse of the disease or condition.The term “treatment” and synonyms are intended to give a therapeuticallyeffective amount of a compound of the invention to an individual in needthereof.

Within the meaning of the invention, “treatment” also includesprevention of relapse or prevention stages, as well as treatment ofacute or chronic signs, symptoms and/or dysfunction. Treatment cantarget symptoms, for example, to suppress symptoms. It can function in ashort period of time, for a medium period of time, or can be a long-termtreatment, such as in the case of maintenance therapy.

The term “therapeutically effective amount” or “effective amount” asused herein refers to an amount of the active ingredient which, whenadministered by the methods of the present disclosure, is sufficient toeffectively deliver the active ingredient to an individual in needthereof for the treatment of a target condition. In the case of canceror other proliferative disorders, a therapeutically effective amount ofthe agent can reduce undesired cell proliferation, reduce the number ofcancer cells, reduce tumor size; inhibit cancer cell infiltration tosurrounding organs; inhibit tumor metastasis; inhibit tumor growth to acertain extent; reduce Bcl-2/Bcl-xL signaling in targeted cells; and/oralleviate one or more symptoms associated with cancer to a certainextent.

In the method of treatment of the invention, a therapeutically effectiveamount of a Bcl-2/Bcl-xL inhibitor can be administered to the patientbefore, after or simultaneously with the administration of atherapeutically effective amount of the chemotherapeutic agent to theindividual in need thereof, wherein the Bcl-2/Bcl-xL inhibitor and thechemotherapeutic agent can each be combined with a pharmaceuticallyacceptable carrier.

In the method of treatment of the invention, the Bcl-2/Bcl-xL inhibitorand the chemotherapeutic agent can be administered together as a singleunit dose or separately as multiple unit doses.

In certain embodiments, the Bcl-2/Bcl-xL inhibitor may be administeredin an amount from about 0.005 to about 500 mg/day, preferably from about0.05 to about 250 mg/day, more preferably from about 0.5 to about 100mg/day. In certain embodiments, the Bcl-2/Bcl-xL inhibitor isadministered at a dose from about 10 mg/week to about 1000 mg/week, fromabout 10 mg/week to about 900 mg/week, from about 10 mg/week to about800 mg/Week, about 10 mg/week to about 700 mg/week, about 10 mg/week toabout 640 mg/week, about 10 mg/week to about 600 mg/week, about 10mg/week to about 500 mg/week, about 10 mg/week to about 400 mg/week,about 10 mg/week to about 300 mg/week, about 10 mg/week to about 200mg/week, or about 20 mg/week to about 100 mg/week, for example about 10,15, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800,850, 900, 950, 1000 mg/week. In certain embodiments, the Bcl-2/Bcl-xLinhibitor is administered at a dose of about 0.005, 0.05, 0.5, 5, 10,20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 mg perdose. In certain embodiments, the Bcl-2/Bcl-xL inhibitor is administeredat a frequency of once a week, twice a week, three times a week, fourtimes a week, five times a week, six times a week or seven times a week.

In certain embodiments, the chemotherapeutic agent can be administeredin an amount from 0.005 mg/day to about 5000 mg/day, including about0.005, 0.05, 0.5, 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500,2000, 2500, 3000, 3500, 4000, 4500 or 5000 mg/day.

Pharmaceutical Use

According to a third aspect of the present disclosure, provided hereinis use of a combination of a Bcl-2/Bcl-xL inhibitor and achemotherapeutic agent in the manufacture of a medicament for treatingcancer, wherein the Bcl-2/Bcl-xL inhibitor and the chemotherapeuticagent are respectively as defined above. The cancer is also as definedabove.

The pharmaceutical composition of the present disclosure can beadministered by any suitable route, for example, by oral, buccal,inhalation, sublingual, rectal, vaginal, intracisternal or intrathecalvia lumbar puncture, transurethral, nasal, or transdermal, or parenteral(including intravenous, intramuscular, subcutaneous, intradoronary,intradermal, intramammary, intraperitoneal, intra-articular,intrathecal, posterior ocular, intrapulmonary, and/or surgicalimplantation at specific sites). Parenteral administration can beaccomplished using a needle and syringe or using high pressuretechniques. In certain embodiments, the Bcl-2/Bcl-xL inhibitor and thechemotherapeutic agent can be administered via the same or differentroutes of administration. In certain embodiments, the route ofadministration includes oral, intravenous or subcutaneous injection.

The toxicity and therapeutic efficacy of the pharmaceutical compositionsof the invention can be determined in cell cultures or experimentalanimals by standard pharmaceutical procedures, for example to determinethe maximum tolerated dose (MTD) of each component, which is defined asthe highest dose not causing toxicity in the animal. Therapeutic indexis the dose ratio between the maximum tolerated dose and the dose thatprovides therapeutic effect (e.g., inhibition of tumor growth). Thedosage can vary within this range, depending on the dosage form employedand the route of administration employed.

Therapeutically effective amounts of Bcl-2/Bcl-xL inhibitors andchemotherapeutic agents required for pharmaceutical use will vary withthe nature of the condition being treated, the length of time requiredfor the activity, and the age and condition of the patient. The amountand interval of administration can be adjusted separately to provide aplasma level of the combination of drugs sufficient to maintain thedesired therapeutic effect. The desired dose can be convenientlyadministered in a single dose or in multiple doses at appropriateintervals, for example, one, two, three, four or more sub-doses per day.Multiple doses are often necessary or required. For example, thepharmaceutical composition of the present disclosure can be administeredat the following frequency: 1 dose/day for 2 days, off for 5 days,lasting for 2 weeks; 1 dose/day for 3 days, off for 4 days, lasting for3 weeks; once a week for 2 weeks; once a week for 4 weeks; or any dosageregimen as appropriate. In certain embodiments, the Bcl-2/Bcl-xLinhibitor and the chemotherapeutic agent can be administered together,simultaneously, sequentially or alternately.

The pharmaceutical compositions of the present disclosure are usuallyadministered in admixture with a pharmaceutically acceptable carrierwhich is selected according to the intended route of administration andstandard pharmaceutical practice. Pharmaceutical compositions for use inaccordance with the present disclosure are formulated in a conventionalmanner using one or more pharmaceutically acceptable carriers.

The pharmaceutical compositions of the present disclosure can beprepared, for example, by conventional mixing, dissolving, granulating,tableting, emulsifying, encapsulating, capturing or lyophilizingprocesses. Suitable formulations will depend on the route ofadministration. When administered orally, the composition will usuallybe in the form of a tablet, capsule, powder, solution or elixirs. Whenadministered in the form of a tablet, the composition may additionallycomprise a solid carrier such as gelatin or an adjuvant. Tablets,capsules and powders contain from about 0.01% to about 95% of aBcl-2/Bcl-xL inhibitor by weight. When administered in liquid form, aliquid carrier such as water, an alcohol or an oil of animal orvegetable origin may be added. The composition in liquid form mayfurther comprise a physiological saline solution, glucose or other sugarsolution or glycerin. When administered in liquid form, the compositionscontain from about 0.1% to about 90% of a Bcl-2/Bcl-xL inhibitor byweight.

When a therapeutically effective amount of a pharmaceutical compositionof the invention is administered by intravenous, cutaneous orsubcutaneous injection, the composition is in the form of anon-pyrogenic, parenterally acceptable aqueous solution. Such aparenterally acceptable solution can be prepared with appropriateconsideration of pH, isotonicity, stability, and the like within theskill of the art. Preferred compositions for intravenous, cutaneous orsubcutaneous injection typically contain an isotonic solvent.

Bcl-2/Bcl-xL inhibitors and/or chemotherapeutic agents can be readilycombined with pharmaceutically acceptable carriers well known in theart. Such a carrier allows the active agent to be formulated into atablet, pill, lozenge, capsule, liquid, gel, syrup, ointment,suspension, and the like. The pharmaceutical preparation for oraladministration can be obtained by adding the active ingredients and asolid excipient, grinding the resulting mixture, and after adding asuitable auxiliary agent if necessary, granulating and pressing toobtain a tablet or tablet core. Suitable excipients include, forexample, fillers, cellulose formulations, disintegrants, binders,lubricants, and the like.

Bcl-2/Bcl-xL inhibitors and/or chemotherapeutic agents are formulatedfor parenteral administration, for example by bolus injection orcontinuous infusion. The injectable preparation may be provided in unitdosage form, for example in ampoules or in multi-dose containers, withadded preservatives. The composition may be in a form such as asuspension in an oily or aqueous solvent, solution or emulsion, and maycontain adjuvants such as suspending agents, stabilizers and/ordispersants.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active agents in aqueous form. In addition,suspensions of Bcl-2/Bcl-xL inhibitors and/or chemotherapeutic agentscan be prepared as suitable oily injection suspensions. Suitablelipophilic solvents include fatty oils or synthetic fatty acid esters.Aqueous injection suspensions may contain substances which increase theviscosity of the suspension. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompound and allow for the preparation of highly concentrated solutions.Alternatively, the compositions of the present disclosure may be inpowder form for constitution with a suitable solvent such as sterilenon-pyrogenic water prior to use.

Bcl-2/Bcl-xL inhibitors and/or chemotherapeutic agents may also beformulated in rectal compositions, such as suppositories or retentionenemas, for example, containing conventional suppository bases. Inaddition to the aforementioned formulations, Bcl-2/Bcl-xL inhibitorsand/or chemotherapeutic agents can also be formulated as depotpreparations. Such long acting formulations may be administered byimplantation (for example subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, a Bcl-2/Bcl-xL inhibitorand/or a chemotherapeutic agent can be formulated with a suitablepolymeric or hydrophobic material (e.g., as an emulsion in an acceptableoil) or an ion exchange resin.

In particular, the Bcl-2/Bcl-xL inhibitor and/or chemotherapeutic agentmay be in the form of a tablet containing an excipient such as starch orlactose, or in the form of a capsule mixed with an excipient, or in theform of an elixir or suspension containing a flavoring agent or acolorant, for oral, buccal or sublingual administration. The aboveliquid preparations may be formulated together with a pharmaceuticallyacceptable additive such as a suspending agent. Bcl-2/Bcl-xL inhibitorsand/or chemotherapeutic agents can also be administered parenterally,such as intravenously, intramuscularly, subcutaneously orintracoronally. For parenteral administration, it is preferably used inthe form of a sterile aqueous solution which may contain othersubstances such as salts or monosaccharides such as mannitol or glucoseto render the solution isotonic with blood.

Anticancer Effect of Bcl-2/Bcl-xL In Vivo

In another aspect, the present application also provides a method fortreating cancer in a patient in need thereof by administering to thepatient an effective amount of a Bcl-2/Bcl-xL inhibitor (e.g., Compound15 or Compound 31 or a pharmaceutically acceptable salt thereof). Incertain embodiments, the Bcl-2/Bcl-xL inhibitor is a single agent. Incertain embodiments, the Bcl-2/Bcl-xL inhibitor acts as an agent incombination with other therapeutic agents, such as chemotherapeuticagents, for combination therapy.

In certain embodiments, the Bcl-2/Bcl-xL inhibitor is Compound 15 or apharmaceutically acceptable salt thereof.

In certain embodiments, the cancer is a solid tumor. In certainembodiments, the cancer is metastatic solid tumor. A “solid tumor” asused herein refers to an abnormal mass of tissue that generally does notcontain cysts or liquid areas. Different types of solid tumors areusually named for the type of cells that form them. A “metastatic” solidtumor as used herein refers to a solid tumor that has spread from theprimary site of origin to a different or a secondary site within thepatient's body.

In some embodiment, the cancer is selected from small cell lung cancer(SCLC), prostate cancer (e.g., prostate adenocarcinoma), ovarian cancer(e.g., epithelial ovarian tumor, ovarian germ cell tumor, ovarianstromal tumor), gastric cancer (e.g., gastric adenocarcinoma),pancreatic cancer (e.g., exocrine cancer, or ampullary cancer), atypicalthymoma, breast cancer (e.g., ductal carcinoma in situ, invasive breastcarcinoma, phyllodes tumor, or breast angiosarcoma), non-small cell lungcancer (NSCLC, e.g., lung adenocarcinoma, squamous cell lung carcinoma,or large cell lung carcinoma), melanoma, colon cancer and brain cancer(e.g., glioma, astrocytoma, oligodendroglioma, ependymoma, meningioma,medulloblastoma, ganglioglioma, Schwannoma, or craniopharyngioma).

In certain embodiments, the patient is relapsed from or refractory to aprior treatment. A “refractory” patient as used herein refers to acancer patient who is resistant at the beginning of the treatment, orbecomes resistant during the treatment. A “relapsed” patient as usedherein refers to a patient who exhibits evidence of cancer progressionafter a period of improvement (such as partial remission or completeremission). The prior treatment can be surgery, chemotherapy, radiotherapy, targeted therapy, immunotherapy, or any combination thereof.

In certain embodiments, the method for treating cancer comprisesadministering to a patient in need thereof a therapeutically effectiveamount of a Bcl-2/Bcl-xL inhibitor, such as Compound 15 or Compound 31,wherein the method comprises at least one 28-day treatment cycle whereinthe Bcl-2/Bcl-xL inhibitor is administered by intravenous infusion twicea week (e.g., Compound 15 or Compound 31 administered by intravenousinfusion over 30 minutes on days 1, 4, 8, 11, 15, 18 and 22 of eachtreatment cycle, 28 days for one treatment cycle) or once a week (e.g.,Compound 15 or Compound 31 administered by intravenous infusion over 30minutes on days 1, 8, 15, and 22 of the treatment cycle, 28 days for onetreatment cycle) for 4 consecutive weeks. The treatment cycle can berepeated as many times as needed (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,or 30 cycles). The therapeutically effective amount is from about 10 mgto about 400 mg of a Bcl-2/Bcl-xL inhibitor.

In some embodiments, the therapeutically effective amount of theBcl-2/Bcl-xL inhibitor is about 20 mg.

In some embodiments, the therapeutically effective amount of theBcl-2/Bcl-xL inhibitor is about 40 mg.

In some embodiments, the therapeutically effective amount of theBcl-2/Bcl-xL inhibitor is about 80 mg.

In some embodiments, the therapeutically effective amount of theBcl-2/Bcl-xL inhibitor is about 120 mg.

In some embodiments, the therapeutically effective amount of theBcl-2/Bcl-xL inhibitor is about 160 mg.

In some embodiments, the therapeutically effective amount of theBcl-2/Bcl-xL inhibitor is about 200 mg.

In some embodiments, the therapeutically effective amount of theBcl-2/Bcl-xL inhibitor is about 240 mg.

In some embodiments, the therapeutically effective amount of theBcl-2/Bcl-xL inhibitor is about 280 mg.

In some embodiments, the therapeutically effective amount of theBcl-2/Bcl-xL inhibitor is about 320 mg.

In some embodiments, the therapeutically effective amount of theBcl-2/Bcl-xL inhibitor is about 360 mg.

In some embodiments, the therapeutically effective amount of theBcl-2/Bcl-xL inhibitor is about 400 mg.

EXAMPLES

The concept of the present disclosure and the technical effects producedby the present disclosure will be further described in conjunction withthe embodiments, so that those skilled in the art can fully understandthe objects, features and effects of the present disclosure. It isunderstood that the following examples are merely illustrative and arenot intended to limit the scope of the invention.

Synthesis of Bcl-2/Bcl-xL Inhibitor

Compound 15:(R)-2-(1-(3-(4-(N-(4-(4-(3-(2-(4-chlorophenyl)-1-isopropyl-5-methyl-4-(methylsulfonyl)-1H-pyrrol-3-yl)-5-fluorophenyl)piperazin-1-yl)phenyl)sulfamoyl)-2-(trifluoromethylsulfonyl)phenylamino)-4-(phenylthio)butyl)piperidine-4-carbonyloxy)ethylphosphonic acid. The method for synthesizing Compound 15 can beprepared by the following method by referring to the description in thespecification of WO2014/113413A.

¹H NMR (300 MHz, CD₃OD): δ 7.93 (d, J=1.9 Hz, 1H), 7.72 (dd, J=9.2, 1.8Hz, 1H), 7.30-7.12 (m, 12H), 6.83-6.42 (m, 5H), 4.46-4.33 (m, 3H), 3.96(s, 1H), 3.54-2.93 (m, 16H), 2.82 (s, 3H), 2.72 (s, 3H), 2.71-2.55 (m,1H), 2.24-1.65 (m, 8H), 1.41 (d, J=7.1 Hz, 6H). MS (ESI): m/z 1268.58(M+H)⁺.

Compound 31:(R)-1-(3-(4-(N-(4-(4-(3-(2-(4-chlorophenyl)-1)-isopropyl-5-methyl-4-(methylsulfonyl)-1H-pyrrol-3-yl)-5-fluorophenyl)piperazin-1-yl)phenyl)sulfamoyl)-2-(trifluoromethylsulfonyl)phenylamino)-4-(phenylthio)butyl)piperidine-4-carboxylic acid. The method for synthesizing Compound15 can be prepared by the following method by referring to thedescription in the specification of WO2014/113413A.

1H NMR (400 MHz, DMSO-d6) δ 9.84 (s, 1H), 7.82 (s, 1H), 7.65 (d, J=8.7Hz, 1H), 7.38 (d, J=8.5 Hz, 2H), 7.32-7.14 (m, 7H), 7.11-6.81 (m, 6H),6.63-6.47 (m, 2H), 6.43-6.30 (m, 1H), 4.33 (p, J=7.1 Hz, 1H), 4.07 (s,1H), 3.32-3.22 (m, 4H), 3.19-3.03 (m, 9H), 2.89 (s, 4H), 2.67 (s, 4H),2.31-1.55 (m, 8H), 1.35 (d, J=7.0 Hz, 6H).

WST Experiment

Cell plating: Anti-proliferation effects were detected by a CCK-8 (CellCounting Kit-8) assay based on water soluble tetrazolium salt (WST).

The cells were seeded in 96-well plates. 95 μL complete medium was addedto each negative control group, and 95 μL cell suspension in completemedium was added at a density of 5-10×10⁴ cells/well to each well to betested.

The MGC80-3 and SW620 used in the examples were purchased from the CellBank of the Shanghai Institute of Biochemistry and Cell Biology, ChineseAcademy of Sciences. MDA-MB-231, PC-3, and NCI-H146 were available fromthe American Type Culture Collection (ATCC) under accession Nos. HTB-26,CRL-1435, and HTB-173, respectively. It should be noted that the cellsused in the present disclosure are all available from ATCC. The cultureconditions of MGC80-3, PC-3, and NCI-H146 were RPMI 1640 medium, 10%fetal bovine serum and 1% dual antibiotics solution. The cultureconditions of MDA-MB-231 were minimal essential medium (containing 0.1mM non-essential amino acids and 1.0 mM sodium pyruvate), 10% fetalbovine serum and 1% dual antibiotics solution. The culture conditions ofSW620 were Leibovitz's L-15 medium, 10% fetal bovine serum and 1% dualantibiotics solution.

Treatment (light protection): In the 96-well culture plate, consideringthat different cells had different sensitivity to drugs, the highestconcentration was selected as 10 μM, and 9 concentrations were obtainedby 1:3 serial dilutions. 5 μL of compound was added to each well andeach concentration was prepared in 2-3 replicate wells. The 96-wellplates were incubated in a 5% CO₂ incubator at 37° C. for 72 hours afterthe addition of the testing compound as different concentrations. Thesynergy of the drug with Compound 15 was tested by the treatment withthe drug (e.g. docetaxel) at 9 different concentrations and Compound 15at 3 fixed concentrations for 72 hours. Each concentration was tested intriplicates.

Reading: At the end of the culture, for adherent cells, the old solutionin the test wells was removed and 100 μl/well CCK-8 test solution (thecorresponding medium containing 10% CCK-8, 5% FBS) was added. Forsuspension cells, CCK-8 stock solution was added directly at 20 μl/well.The plates were incubated at 37° C. in a CO₂ incubator for 2-4 hours.

The OD value was measured using a microplate reader (SpectraMax Plus384, Molecular Devices, LLC., US) A450 nm. Mean OD value was taken fromtriplicate wells. The percentage of cell viability was calculated by thefollowing formula:(O.D. test well−O.D. blank control well)/(O.D. cell control well−O.D.blank control well)×100%.

The IC₅₀ was calculated using the nonlinear regression data analysismethod of Graphpad Prism 6.0 software. The results are shown in FIG. 1and Table 2.

For the combination study, the mean OD value of the triplicate wells ofthe single drug control was normalized to calculate cell viability. Thesynergistic effect of the two compounds was determined by comparing theIC50 of the combination curve with the single drug curve (thecombination curve shifted to the left).

Evaluation Method of In Vivo Pharmacodynamic Study

Subcutaneous xenograft tumor model of human cancer was established inimmunodeficient mouse by cell inoculation: tumor cells in logarithmicgrowth phase were collected, counted, and then re-suspended in 1×PBS.The concentration of the cell suspension was adjusted to 2.5-5×10⁷/mL.Tumor cells were inoculated subcutaneously in the right back of theimmunized normal mouse with a 1 mL syringe (4 gauge needle) at5-10×10⁶/0.2 mL/mouse. Relevant parameters were calculated withreference to China's NMPA “Guidelines for Non-clinical ResearchTechniques of Cytotoxic Antitumor Drugs”.

Animal weight and tumor size were measured twice a week during thestudy. The animals were checked daily for morbility and etc. At the timeof routine monitoring, the animal were checked for any effects of tumorgrowth and treatments on behaviors such as mobility, food and waterconsumption, body weight gain/loss, eyes, hairing and any otherabnormalities. Mortality and clinical signs observed during the studywere recorded in the raw data. The dosing of the drugs and themeasurement of mouse weight and tumor volume were all conducted in alaminar flow cabinet. According to requirements of the study protocol,plasma and tumor tissue were collected, weighed and photographed at theend of last dosing. Plasma and tumor samples were frozen at −80° C. forlater use.

Tumor volume (TV) was calculated as:TV=a×b ²/2wherein a and b represent the measured length and width of tumor,respectively.

Relative tumor volume (RTV) was calculated as:RTV=V _(t) /V ₁wherein V₁ was the tumor volume on the first day of dosing, and V_(t)was the tumor volume measured on day t after dosing.

Relative tumor proliferation rate T/C (%), as an indication ofanti-tumor effectiveness, was calculated as:Relative tumor proliferation rate T/C (%)=(T _(RTV) /C _(RTV))×100%,wherein T_(RTV) was RTV in treatment group, and C_(RTV) was RTV invehicle control group.

Tumor remission rate (%) was calculated by dividing the number of tumorbearing mice that show SD (stable disease), PR (partial regression) andCR (complete regression) after treatment by the total number of mice inthe group×100%.Change of body weight (%)=(measured body weight−body weight atrandomization)/body weight at randomization×100%.

Efficacy evaluation criteria: according to China's NMPA “Guidelines forNon-clinical Research Techniques of Cytotoxic Antitumor Drugs” (November2006), T/C (%) value ≤40% and p<0.05 by statistical analysis wereconsidered as efficacious. A weight loss of mice greater than 20%, orgreater than 20% drug death are considered to indicate an excessivelytoxic dosage.

In Vitro Results

As shown in FIG. 1 and Table 2, in MDA-MB-231 triple-negative breastcancer tumor cell line, combination therapy using Compound 15 anddocetaxel (TXT) demonstrated a stronger inhibition effect on tumor cellproliferation, as indicated by a combination curve shifted to left. TheIC50 value of the combination therapy group was lower than the IC50 ofeach monotherapy group.

TABLE 2 Results of Compound 15 and docetaxel administered in combinationor alone IC₅₀ IC₅₀ IC₅₀ Component Component Cell (A (B (combination A Bline alone) alone) of A + B) Compound docetaxel MDA- 2.599/ 2.77e−5/9.37e−6/ 15 MB- 4.554 7.87e−5 1.62e−5 231 μM μM μM

In Vivo Test

Example 1. Combinatory Anti-Cancer Effect of Compound 15 in Combinationwith Docetaxel in Breast Cancer Xenograft Model of Mice Bearing HumanMDA-MB-231 Cell Line

MDA-MB-231 is a model of human triple-negative breast cancer. This studyevaluated the anti-cancer therapeutic effect of Compound 15 incombination with docetaxel in the xenograft model. As shown in FIG. 2,Compound 15 (100 mg/kg) enhanced the anti-cancer effect of docetaxel asa single agent. Complete regression was observed in ⅛ of the tumors atthe end of dosing and lasted until the end of the study in the groupdosed with Compound 15 (100 mg/kg) and docetaxel. The study results showthat a more significant anti-cancer effect was achieved intriple-negative breast cancer when Compound 15 and docetaxel wereadministered in combination than alone.

Example 2. Combinatory Anti-Cancer Effect of Compound 15 in Combinationwith Docetaxel in Xenograft Model of Mice Bearing Human NCI-HI146 CellLine Small Cell Lung Cancer

This study investigated the anti-cancer therapeutic effect of Compound15 in combination with docetaxel in the human NCI-H146 small cell lungcancer model. As shown in FIG. 3, Compound 15 (25 mg/kg) enhanced theanti-cancer effect of docetaxel as a single agent. On day 4 of dosing,in the group administered with Compound 15 (25 mg/kg) and docetaxel,partial tumor regression was observed in 1 out of the 7 study animals,and after three weeks, complete tumor regression was observed in 5 outof the 7 study animals. The study results show that a more significantanti-cancer effect was achieved in small cell lung cancer when Compound15 and docetaxel were administered in combination than alone.

Example 3. Combinatory Anti-Cancer Effect of Compound 15 in Combinationwith Docetaxel in Xenograft Model of Mice Bearing Human PC3 Cell LineProstate Cancer

This study investigated the anti-cancer therapeutic effect of Compound15 and docetaxel in the PC3 human prostate cancer model. As shown inFIG. 4, Compound 15 (100 mg/kg) enhanced the anti-cancer effect ofdocetaxel as a single agent. Compound 15 (100 mg/kg) alone showed aminor anti-cancer effect with T/C (%) of 84.4% (P=0.4665). Docetaxel (8mg/kg) alone also showed a minor anti-cancer effect with T/C (%) of72.4% (P=0.1579). Compound 15 combined with docetaxel showed betteranti-cancer effect than the single drug with T/C (%) of 51.5%(P=0.0209). The study results showed that a more significant anti-cancereffect was achieved in prostate cancer when Compound 15 and docetaxelwere administered in combination than alone.

Example 4. Combinatory Anti-Cancer Effect of Compound 15 in Combinationwith Docetaxel or Cisplatin in Xenograft Model of Mice Bearing HumanMGC80-3 Cell Line Gastric Cancer

This study investigated the anti-cancer therapeutic effect of Compound15 with docetaxel or cisplatin in the MGC80-3 human gastric cancermodel. As shown in FIG. 5, Compound 15 (100 mg/kg) enhanced theanti-cancer effect of docetaxel as a single agent. Docetaxel (8 mg/kg)alone showed a minor anti-cancer effect with T/C (%) of 54.5%(P=0.6650). Compound 15 combined with docetaxel showed a moresignificant anti-cancer effect than docetaxel alone with T/C (%) of47.8% (P=0.0572).

As shown in FIG. 6, Compound 15 (100 mg/kg) increased the anti-cancereffect of docetaxel or cisplatin as a single agent. Docetaxel (12 mg/kg)alone showed a minor anti-cancer effect with T/C (%) of 39.0%(P=0.0003). Compound 15 combined with docetaxel showed more significantanti-cancer effect than docetaxel alone with T/C (%) of 7.8% (P<0.0001),and complete tumor regression was observed in 2 out the 7 study animalsat the third week of dosing. The group treated with cisplatin (3 mg/kg)alone showed T/C (%) of 65.0% (P=0.0034) at the end of dosing, and thegroup treated with Compound 15 and cisplatin showed T/C (%) of 53.6%(P=0.0001).

The above study results show that a more significant anti-cancer effectwas achieved in gastric cancer when Compound 15 and docetaxel orcisplatin were administered in combination than alone.

Example 5. Combined Anti-Cancer Effect of Compound 15 in Combinationwith Irinotecan in Xenograft Model of Mice Bearing Human SW620 Cell LineColon Cancer

This study investigated the anti-cancer therapeutic effect of Compound15 and irinotecan in the SW620 human colon cancer model. As shown inFIG. 7, Compound 15 (100 mg/kg) enhanced the anti-cancer effect ofirinotecan alone. Compound 15 (100 mg/kg) showed a minor anti-cancereffect with T/C (%) of 60.6%; Irinotecan (50 mg/kg) alone also showed aminor anti-cancer effect with T/C (%) of 29.9%; Compound 15 combinedwith irinotecan in a BIW dosing cycle (50 or 100 mg/kg of Compound 15)and in a QW dosing cycle (100 mg/kg of Compound 15) showed T/C (%) of23.5% (P=0.0001), 9.1% (P<0.0001) and 11.4% (P=0.0001), respectively.The results of this study indicate that a more significant anti-cancereffect was achieved in prostate cancer when Compound 15 and irinotecanwere administered in combination than alone.

As seen from the above studies where Compound 15 was used as a singleagent or combined with chemotherapeutic agents for in vitro cellanti-proliferation assay, the inhibition on proliferation in a series oftumor cell lines was enhanced when Compound 15 was combined withchemotherapeutic agents.

The results of the studies, where Compound 15 was combined withchemotherapeutic agents in NCI-H146 human small cell lung cancer model,MDA-MB-231 human triple-negative breast cancer model, PC-3 humanprostate cancer model, MGC80-3 human gastric cancer model and SW620human colon cancer model, showed that Compound 15 can enhance theanti-cancer effect of chemotherapy agents.

These results showed that the combination of Bcl-2/Bcl-xL inhibitorswith chemotherapeutic agents had great potential for cancer treatment.

Example 6. Compound 15 for Use in the Treatment of Patients withMetastatic Solid Tumors

Compound 15 is a novel dual Bcl-2/Bcl-xL inhibitor. Embodying a uniquedevelopment strategy, it artfully reduces targeted platelet toxicitywhile maintaining strong in vivo anti-cancer activity. Compound 15inhibits tumor growth in a human cancer xenograft model while showingsignificantly less thrombocytopenia.

Three clinical trials (Phase I or Phase I/II) with similar study designswere conducted in the United States, Australia, and China. During thedose escalation phase, the patient received Compound 15 (10-400 mg)twice weekly or weekly intravenously for a 28-day treatment cycle. Tumorresponse assessment was performed every 2 cycles according to RECIST 1.1criteria. The purpose of the study was to assess safety, maximumtolerated dose (MTD)/recommended phase II dose (RP2D), pharmacokinetics(PK), pharmacodynamics (PD), and the preliminary efficacy of Compound 15as a single agent in metastatic solid tumors.

1) Pooled Results as of Jan. 31, 2019

As of Jan. 31, 2019, 44 patients (US: 21 patients, China: 13 patients,Australia: 10 patients) received treatment with 7 different dose levelsof Compound 15. The dose level currently under study is 320 mg twice aweek.

A pooled analysis of the study showed that Compound 15 was welltolerated at all tested dose levels. The MTD had not been reached. Mostadverse events (AEs) were grade 1 or 2 and no drug-related AEs thatcaused drug discontinuation were found. The most common grade 3/4 AEswere hyponatremia (11.4%), decreased lymphocyte count (6.8%), andelevated lipase (6.8%).

19 patients with small cell lung cancer (SCLC) underwent at least onepost-treatment tumor evaluation, and 1 of the US patient had partialremission (PR) at a dose of 40 mg. The partial remission lasted for along time and the patient had been treated with Compound 15 for morethan 20 courses. 4 Chinese patients achieved stable disease (SD) at adose of 80 mg/240 mg, and two of them were stable for more than fourcourses. In addition, 5 cases of stable disease in other tumor types(e.g., CRC) were reported in Australian studies.

PK analysis showed that AUC and Cmax on day 1 increased proportionallywith dose in the dose range of 10-160 mg, and AUC and Cmax werecomparable between US and Chinese patients at the same dose level.

This study showed that Compound 15 was well tolerated at all tested doselevels. Dose escalation is continued in a twice-weekly and weekly dosingschedule. A preliminary anti-cancer activity of single agent wasobserved during dose escalation.

2) Results of the U.S. Trial as of Dec. 21, 2019

As of Dec. 21, 2019, 42 US patients with metastatic solid tumors (TableA) received Compound 15 treatment ranging from 10-400 mg in at least one28-day cycle. The patients were treated in 2 cohorts. 31 patients incohort 1 received Compound 15 twice a week (BIW) by intravenous (IV)infusion at a dose of 10 mg, 20 mg, 40 mg, 80 mg, 160 mg, 240 mg, 320mg, or 400 mg; 11 patients in cohort 2 received Compound 15 once a week(QW) by IV infusion at a dose of 240 mg or 320 mg.

TABLE A Primary Cancer, n (%) Compound 15 (N = 42) SCLC 8 (19.0%)Ovarian Cancer 5 (11.9%) Colon Cancer 5 (11.9%) Pancreatic Cancer 4(9.5%) NSCLC 4 (9.5%) Breast Cancer 2 (4.7%) Others (Brain, skin, 14(33.3%) prostate, etc.)

4 DLTs (grade 4 thrombocytopenia) were observed in 4 patients at 320 mgand 400 mg BIW, respectively. Rapid platelet drop was observed inpatients at these 2 dose levels, which was transient and resolvedrapidly within 2-6 days without any medical intervention (FIG. 33(a)).Most adverse events (AEs) were grade 1 or 2 (G1 or G2), and 26.2%patients had ≥G3 treatment emergent adverse events (TRAEs). The mostcommon TRAEs were platelet count decreased (14.3%), aspartateaminotransferase increased (9.5%), and alanine aminotransferaseincreased (7.1%).

Compound 15 was well-tolerated up to 240 mg. MTD/RP2D was determined as240 mg QW.

Among these 42 patients, post-baseline tumor measurements were obtainedfrom 36 patients. 3 subjects were just starting the treatment of Cycle 2by the data cut-off date, 2 patients were replaced in Cycle 1, and 1patient discontinued treatment for radiotherapy. Encouraging anti-tumoreffect has been observed in this first-in-human study.

Partial response (PR) was observed in 3 patients, including: a 60 yearsold male patient (patient #002-001) with SCLC, who hadrelapsed/progressed disease after previous treatment of Carboplatin,Etoposide, Cisplatin and Nivoluma, having PR lasted for 19 cycles (doselevel: 40 mg, BIW); a 59 years old male patient (patient #001-016) withprostate cancer (neuroendocrine and adenocarcinoma), who had receivedsurgery, chemotherapy and checkpoint inhibitor treatment prior to theenrollment, having PR result at his 1st tumor assessment; and a 62 yearsold female patient (patient #002-001) with ovarian cancer, whopreviously had been treated with surgery followed by chemotherapy andtargeted therapy (FIGS. 33(b) and 33(c)).

Stable disease (SD) was observed in 7 patients, including 4 patients(patients #001-001, 001-003, 002-004 and 002-034) at 10 mg (BIW), 20 mg(BIW), 40 mg (BIW) and 240 mg (BIW), respectively, and 3 patients(patients #001-014, 001-018 and 001-019) from at a dose of 320 mg fromcohorts 1 and 2. Among these 7 patients, 5 patients had SD lasted formore than 4 cycles (including 2 patients had SD lasted for more than 6cycles) (FIG. 33(c))

PK analysis showed that C_(max) and AUC were approximately doseproportional over the range of 10 mg to 400 mg following the IV infusionon Day 1 (FIG. 33(d)). No significant accumulation was observed withonce or twice weekly dosing.

In conclusion, 42 patients (31 on BIW and 11 on QW) with metastaticsolid tumors received Compound 15 treatment ranging from 10-400 mg in a28-day cycle. The most common Grade ≥3 AEs was thrombocytopenia, whichwas the on-target toxicity and shown to be transient and resolvedrapidly within 2-6 days. Four DLTs (Grade 4 thrombocytopenia) werejudged by investigators at 400 mg and 320 mg. Compound 15 waswell-tolerated up to 240 mg. MTD/RP2D was determined as 240 mg. Compound15 displayed linear PK and was approximately dose proportional over thedose range of 10-400 mg. Encouraging anti-tumor effect has been observedin this first-in-human study, which supports further development ofPalcitocalx in combination therapies for solid tumors.

Example 7. Therapeutic Efficacy of Compound 15 in Lung Cancer XenograftModel

The therapeutic efficacy of compound 15 alone and its multiplecombinations in the treatment of subcutaneous HuPrime® Lung cancerxenograft model LU5250 and LU5188 in female NOD.SCID mice was evaluated.

Either LU5250 or LU5188 tumor mass with rough size of 3×3×3 mm³ wereinoculated into female NOD.SCID mice and the day of randomization wasdenoted as day 1. The treatment was initiated on day 1 when tumorsapproximated a mean volume 136 mm³ of LU5250 and 155 mm³ of LU5188,respectively. There were 6 groups enrolled in each model eventually. SeeTable 3 for the mode of administration and doses. Both models were takendown on day 21.

TABLE 3 Dose Dosing Dosing Dosing level solution volume Route offrequency & Group No. Treatment (mg/kg) (mg/ml) (μl/g) dosing duration 12 Compound — — 10 i.v. BIW × 7 15 Vehicle doses Irinotecan 10 i.p. Q4D ×3 w Vehicle 2 2 Compound 50 5 10 i.v. BIW × 7 15 doses 3 2 Irinotecan 101 10 i.p. Q4D × 3 w 4 2 Compound 50 5 10 i.v. BIW × 7 15 dosesIrinotecan 10 1 10 i.p. Q4D × 3 w 5 2 Paclitaxel 10 1 10 i.v. Q4D × 3 w6 2 Compound 50 5 10 i.v. BIW × 7 15 doses Paclitaxel 10 1 10 i.v. Q4D ×3 w Note: a. No.: animal number per group. b. Dosing Volume was adjustedaccording to individual body weight (BW). c. The day of randomizationwas set as day1. The day of treatment was set as day1. d. For BIW,treatment was administered on day1, day4, day8, day11, day15, day18 andday21; For Q4D, treatment was administered on day1, day5, day9, day13,day17, day21. e. For combo, Compound 15 was dosed first, andIrinotecan/Paclitaxel was administered 1 hour after. f. If mousesuffered a body weight loss (BWL) >20%, it must be removed andeuthanized.

Tumor volumes were measured twice weekly in two dimensions using acaliper, and the volume was expressed in mm³ using the formula: V=0.5a×b² where a and b are the long and short perpendicular diameters of thetumor, respectively. Dosing as well as tumor and body weight measurementwere conducted in a Laminar Flow Cabinet. Relative tumor volume (RTV)was calculated using the following formula: RTV=Vt/V1 where V1 and Vtare the average tumor volumes on the first day of treatment (day 1) andthe average tumor volumes on a certain time point (day t). Synergy Ratiowas calculated using the following formula (Clarke R., Breast CancerResearch & Treatment, 1997, 46 (2-3):255-278.7): SynergyRatio=((A/C)×(B/C))/(AB/C); wherein A is response to treatment A; B isresponse to treatment B; C is response to vehicle control; AB: responseto combination of treatment A and B.

Standard NCI procedures were used to calculate tumor parameters.Relative tumor proliferation rate (% T/C) was calculated as the mean RTVof treated tumors (T) divided by the mean RTV of control tumors(C)×100%. The percentage T/C value is an indication of antitumoreffectiveness: a value of T/C<42% is considered significant antitumoractivity by the NCI. A T/C value <10% is considered to indicate highlysignificant antitumor activity, and is the level used by the NCI tojustify a clinical trial if toxicity and certain other requirements aremet (termed DN-2 level activity). A body weight loss (mean of group) ofgreater than 20%, or greater than 20% of drug deaths are considered toindicate an excessively toxic dosage.

The tumor growth curves (mean tumor volume over time) of LU5250 andLU5188 are shown in FIGS. 8(a), 8(b), 9(a) and 9(b), respectively. Thetumor growth inhibition is summarized in Table 4 and Table 5 below. Theresults of mean body weight changes of LU5250 and LU5188 are shown inFIGS. 10(a), 10(b), 11(a) and 11(b), respectively.

TABLE 4 Antitumor activity of Compound 15 combined with Paclitaxel orIrinotecan in SCLC patient-derived xenograft LU5250 Synergy RTV @ T/C(%) Ratio @ Group Treatment day21 @ day 21 day 21 1 vehicle 7.13 ± 0.78— — 2 Compound 15 9.75 ± 3.77 136.8 — 3 Irinotecan 1.92 ± 0.24 26.9 — 4Compound 15 + 1.60 ± 0.24 22.5 1.63 Irinotecan 5 Paclitaxel 2.33 ± 0.1332.6 — 6 Compound 15 + 1.71 ± 0.43 24.0 1.86 Paclitaxel Ratio > 1,synergistic; ratio = 1, additive; ratio < 1, antagonistic Note: 1,Relative tumor volume (RTV): mean ± SEM. 2, Relative tumor proliferationrate (% T/C) = mean RTV of treated tumors (T_(RTV))/mean RTV of controltumors (C_(RTV)) × 100%

TABLE 5 Antitumor activity of Compound 15 combined with Paclitaxel orIrinotecan in SCLC patient-derived xenograft LU5188 Synergy RTV @ T/C(%) @ Ratio @ Group Treatment day 21 day 21 day 21 1 vehicle 13.60 ±1.70 — — 2 Compound 15 17.05 ± 6.59 125.4 — 3 Irinotecan  8.41 ± 2.4961.8 — 4 Compound 15 +  7.47 ± 1.33 55.0 1.41 Irinotecan 5 Paclitaxel20.55 ± 3.20 151.1 — 6 Compound 15 +  8.78 ± 0.50 64.6 2.94 PaclitaxelRatio > 1, synergistic; ratio = 1, additive; ratio < 1, antagonisticNote: 1, Relative tumor volume (RTV): Mean ± SEM. 2, Relative tumorproliferation rate (% T/C) = mean RTV of treated tumors (T_(RTV))/ meanRTV of control tumors (C_(RTV)) × 100%

Example 8. Therapeutic Efficacy of Compound 15 in Lung Cancer XenograftModel

The therapeutic efficacy of compound 15 alone and in multiplecombinations in the treatment of subcutaneous NCI-H146 lung cancerxenograft model was evaluated.

1×10⁷ NCI-H146 tumor cells were pre-treated in 0.1 mL of phosphatebuffer and Matrigel (7:3) and inoculated into female SCID mice. The dayof randomization was denoted as day 1. The treatment was initiated onday 1. The study was conducted in 8 groups of mice. See Table 6 for themode of administration and doses. The entire study was taken down on day21. The study was conducted in 40 mice. All the animals were randomizedinto 8 experimental groups. Tumors approximated a mean volume 192 mm³ atrandomization.

TABLE 6 Dose Dosing Dosing Dosing level solution volume Route offrequency & Group No. Treatment (mg/kg) (mg/ml) (μl/g) dosing duration 15 Vehicle — — 10 i.v. BIW × 7 (Compound doses 15 vehicle) 10 i.v. QW ×(Paclitaxel 3 wks vehicle) 2 5 Compound 20 2 10 i.v. BIW × 7 15 doses 35 Irinotecan 5 0.5 10 i.p. QW × 3 wks 4 5 Paclitaxel 10 1 10 i.v. QW × 3wks 5 5 Cisplatin 1 0.1 10 i.p. Q4D × 3 wks 6 5 Compound 20 2 10 i.v.BIW × 7 15 doses Paclitaxel 10 1 10 i.v. QW × 3 wks 7 5 Compound 20 2 10i.v. BIW × 7 15 doses Irinotecan 5 0.5 10 i.p. QW × 3 wks 8 5 Compound20 2 10 i.v. BIW × 7 15 doses Cisplatin 1 0.1 10 i.p. Q4D × 3 wks Note:a. No.: animal number per group. b. Dosing Volume was adjusted accordingto individual BW. c. The day of randomization was set as day1. The dayof treatment was set as day1. d. For BIW, treatment was administered onday1, day4, day8, day11, day15, day18 and day21; For Q4D, treatment wasadministered on day1, day5, day9, day13 and day17. For QW, on day1, day8and day15. e. For combination, in group 6/7/8, Compound 15 was dosedfirst, and Paclitaxel/Irinotecan/Cisplatin was administered 1 hourafter.

The method of measuring and calculating tumor volume, and the method ofcalculating tumor parameters are the same as those in Example 7. Thetumor growth curves (mean tumor volume over time) of 8 different groupsare shown in FIGS. 12(a), 12(b) and 12(c). The tumor growth inhibitionis summarized in Table 7 below. The results of mean body weight changesin the tumor bearing mice are shown in FIGS. 13(a), 13(b) and 13(c).

TABLE 7 Antitumor activity in the treatment of NCI-H146 model T/ SynergyRTV C(%) Ratio @day @day @day mRECIS mRECIS Group Treatment 21 21 21T_best T_last 1 Vehicle 2.9 ± 0.5 — — 2/5 SD, 5/5 PD 3/5 PD 2 Compound2.0 ± 0.3 70.0 — 4/5 SD, 1/5 SD, 15 1/5 PD 4/5 PD 3 Irinotecan 1.0 ±0.2***^(##) 34.4 — 3/5 PR 1/5 PR, 2/5 SD 3/5 SD, 1/5 PD 4 Compound 0.2 ±0.1*** 5.7 4.2 4/5 PR, 4/5 PR, 15 + 1/5 CR 1/5 CR Irinotecan 5Paclitaxel 1.4 ± 0.4** 48.4 — 3/5 PR, 3/5 SD, 2/5 SD 2/5 PD 6 Compound0.8 ± 0.3*** 27.1 1.2 3/5 PR, 1/5 PR, 15 + 2/5 SD 3/5 SD, Paclitaxel 1/5PD 7 Cisplatin 2.6 ± 0.2 90.2 — 2/5 SD, 5/5 PD 3/5 PD 8 Compound 1.4 ±0.2* 49.5 1.3 1/5 PR, 4/5 SD, 15 + 4/5 SD 1/5 PD Cisplatin *p < 0.05,***p < 0.001 vs. vehicle control group; ^(##)p < 0.01 vs. Compound 15;Ratio > l, synergistic; ratio = 1, additive; ratio < 1, antagonisticNote 1, mRECIST (Gao H, Nature medicine, 2015, 21(11):1318): modifiedResponse Evaluation Criteria in Solid Tumors; mRECIST_best: the bestresponse during the treatment; mRECIST_last: response on the last day oftreatment. 2, PR: partial response, CR: complete response, SD: stabledisease, PD: progressive disease

Example 9. In Vitro Study of Compound 31 in Small Cell Lung Cancer CellLine Model

The study was to test the anti-proliferation and apoptosis-inducingeffects of Compound 31 in combination with chemotherapy agents for smallcell lung cancer in human small cell lung cancer cells. Chemotherapeuticagents used in this study included paclitaxel, cisplatin, etoposide,irinotecan, and topotecan.

The Cell Titer-Glo® luminescent cell viability assay (CTG) andCaspase-Glo® 3/7 assay were used to assess the effects of Compound 31 asa single agent or in combination with other chemotherapeutic agentsagainst proliferation and in induction of apoptosis, respectively.

Cells in the logarithmic growth phase were collected for Cell Titer-Glo®(CTG) luminescence cell viability assay. 10 μL of the diluted drugsolution was added into a 96-well plate while 10 μL of the medium wasadded into the 96-well control plate. For the study of the combination,5 μL of the indicated test reagent was added to each well. At the end ofthe study, the 96-well plates were equilibrated to room temperature, andthen 30 μL of CTG reagent (protected from light) was added to each wellto induce cell lysis. The luminescent signal was then detected using aBiotek Synergy H1 plate reader. Percent cell viability (%) wascalculated using the mean luminescence value (LN) of the replicate wells(blank) of the control group, according to the following formula:percent cell viability (%)=(fluorescence signal value of experimentalcells−fluorescence signal value of negative control cell)/(fluorescencesignal value of control cells−fluorescence signal value of negativecontrol cells)×100%.

Cell viability curve was plotted using Graphpad Prism 6.0 software(Golden software, Golden, Colo., USA). For the study of the drugcombination, the Combination Index (CI) was calculated using CalcuSynsoftware (BIOSOFT, UK) to further analyze the combined effects of theindicated drugs (Chou, Cancer Research 70, 440-446). CalcuSyn is aprofessional analysis software for mixed drug treatment. It can quicklyand accurately calculate various drug interactions, includingsynergistic effect, additive effect and antagonistic effect. If the CIvalue of two drugs is <1, it indicates that the two drugs have asynergistic effect; if the CI value=1, it indicates that the two drugshave an additive effect; if the CI value is >1, it indicates that thetwo drugs have an antagonistic effects.

The assay kit (Promega) in the Caspase-Glo® 3/7 assay was used toquantify the activation of caspase 3/7 during the apoptosis induced bythe drugs or their combinations. The cell seeding and drug dilutionprocedures are the same as those described above. Cells in 96-wellplates were treated with the drugs or their combinations and then heldat room temperature for 30 minutes after equilibration. 30 μL ofCaspase-Glo® 3/7 reagent (protected from light) was added to each welland mixed thoroughly to induce cell lysis. The 96-well plate was held atroom temperature for an additional 30 minutes to stabilize theluminescent signal. Luminescence signals were detected using a BiotekSynergy H1 plate reader. The caspase 3/7 activation curve was plottedusing Graphpad Prism 6.0 software 4.

Small cell lung cancer cell lines were available from the followingsources: (1) ATCC accession No. HTB-119 (NCI H69); (2) ShanghaiInstitute of Biochemistry and Cell Biology (NCI-H446 and NCI-H146).Cells were cultured at 37° C. in a humidified environment containing 5%CO₂.

The purpose of this study was to evaluate the effect of Compound 31 as asingle agent or in combination with other chemotherapy agents (includingpaclitaxel, cisplatin, etoposide, irinotecan and topotecan) ininhibiting cell proliferation/viability in small cell lung cancer cellline NCI-H146, NCI-H69 and NCI-H446. As shown in FIGS. 14(a)-(c),15(a)-(c), 16(a), 16(b), 17(a), 17(b), 18(a), and 18(b), the combinationof Compound 31 with paclitaxel, cisplatin, etoposide, irinotecan ortopotecan has a much greater effect on tumor growth inhibition thanCompound 31 alone or the corresponding chemotherapy agent alone, whichcan be seen from its response curve shifting to the left.

The combination index (CI) can be further calculated based on thedifferent doses of the combinations specified in FIGS. 14(a)-(c),15(a)-(c), 16(a), 16(b), 17(a), 17(b), 18(a), and 18(b). In all celllines tested, the combination of Compound 31 with paclitaxel ortopotecan achieved synergy at lower concentrations, whereas synergy wasobserved with Compound 31 in combination with cisplatin, irinotecan oretoposide at higher concentration.

The results confirmed that in human small cell lung cancer cell line,the combination of Compound 31 with a chemotherapeutic agent can producea synergistic anti-proliferative effect. The original dose-dependentgrowth inhibition curves and combination index are shown in FIGS.14(a)-(c), 15(a)-(c), 16(a), 16(b), 17(a), 17(b), 18(a), and 18(b).

The inventors next tested the effect of Compound 31 as a single agent orin combination with other chemotherapeutic drugs on induction ofapoptosis by measuring caspase 3/7 activation.

The results of the study showed (see FIGS. 19, 20, 21(a), 21(b), 22(a),22(b), 23(a), and 23(b)) that in all cell lines and tested drugs, thedrug treatment increased the concentration of activated (lysed) Caspase3/7 in a dose-dependent manner. The drug combination obtained asignificantly higher concentration than the single agent, indicatingthat the drug combination can induce apoptosis more effectively.Interestingly, in some cases, caspase 3/7 activation is divided into twophases. After an increase in the initial phase (increased by a strongerintensity in the combination group), the activated caspase 3/7concentration reached a plateau and began to decline in the later phase.This two-phase activation of caspase 3/7 is more pronounced in the drugcombination group and occurs more rapidly. This is in line with theprinciple that combination of drugs triggers more effective and fasterapoptosis.

Discussion:

In an in vitro study of small cell lung cancer cell line model, wecombined Compound 15 with standard chemotherapy drugs for small celllung cancer, including paclitaxel, topotecan, cisplatin, etoposide, andirinotecan. These chemotherapies kill cancer cells by inhibiting mitosis(paclitaxel) or adding genotoxicity (other drugs) to cells, therebydown-regulating anti-apoptotic proteins (e.g., Mcl-1 is inhibited bypaclitaxel during mitosis (Huang S, et al. Oncotarget), 7(25))) orup-regulating pro-apoptotic proteins (e.g., NOXA is inhibited by thetopoisomerase 1 inhibitor irinotecan (Okumura K J, et al. CancerResearch, 14 (24))). Compound 15 has a unique mechanism of action. Itcan target intrinsically overexpressed anti-apoptotic proteins whilealso overcoming potential resistance from standard chemotherapy,including up-regulation of Bcl-2, Bcl-xL or Bcl-W (Shi J et al. CancerResearch 71 (13); Bah N et al. Cell Death and ‘Disease, 5, e1291; doi:10.1038/cddis.2014.251; Huang S, et al. Oncotarget, 7(25)).

The study shows that Compound 31 as a single agent can inhibitproliferation of small cell lung cancer cells. Apoptosis is induced byinducing activation of caspase 3/7. The combination of Compound 15 withother chemotherapeutic drugs can synergistically enhance inhibition andmay have stronger anti-tumor activity in vivo. Therefore, thecombinations of Compound 31 with standard chemotherapeutic drugs havegreat therapeutic potential for small cell lung cancer and warrantfurther clinical trials.

Example 10. Compound 15 for Patients with EGFR Inhibitor Resistance

The therapeutic effect of Compound 15 in combination with cisplatin ordocetaxel on the subcutaneous NCI-H1975-L858R-T790M-C797S NSCLCxenograft model was evaluated.

Osimertinib (AZD9291) is a 3rd generation EGFR inhibitor developed toovercome resistance arising from earlier tyrosine kinase inhibitors(TKIs) therapies, typically associated with the occurrence of T790Mmutation. However, most patients experience disease progression after1-2 years on the targeted therapy due to de novo genomic abnormality,such as C797S mutation or exon 20 insertion in EGFR gene. How to killthese newly acquired resistance cells becomes an emergent challenge inthe clinic (Nagano, 2018, “Mechanism of Resistance to Epidermal GrowthFactor Receptor-Tyrosine Kinase Inhibitors and a Potential TreatmentStrategy”, Cell 15; 7 (11).pii E212).

The study tested if the combination of chemotherapies and Compound 15was effective in osimertinib resistant H1975 EGFR C797S xenograft. Thecombination showed a synergistic inhibition in tumor progression inH1975 CDX model carrying EGFR L858R/T790M/C797S mutations.

6 groups of NOD/SCID mice bearing NCI-H1975 (EGFR L858R/T790M/C797S)tumors were treated with vehicle, Compound 15, docetaxel (TXT),cisplatin, Compound 15 in combination with docetaxel or Compound 15combination with cisplatin. Compound 15 was administered intravenouslyat a dose of 100 mg/kg twice a week for a total of 5 doses. Cisplatinwas administered intravenously at a dose of 5 mg/kg once a week for atotal of 3 doses. Docetaxel was administered intravenously at a dose of8 mg/kg once a week for a total of 2 doses. When administered incombination, the drugs are each administered at the doses and frequencyof administration when administered alone. The treatment with TXT as thesingle agent showed antitumor activity with T/C value of 21.4% at day22. The combination treatment of Compound 15 and docetaxel achievedsubstantial antitumor activity with T/C value of 5.3%. Synergy Ratiocalculated as 2.53 (>1) indicates a synergistic combination effect. Thetreatment with cisplatin as the single agent showed moderate antitumoractivity with T/C value of 66.1% at day 22. The combination treatment ofCompound 15 and cisplatin achieved synergistic antitumor activity withT/C value of 26.8%, and Synergy Ratio was 1.55 (>1). The method ofmeasuring and calculating tumor volume, and the method of calculatingtumor parameters are the same as those in Example 7. The tumor growthcurves (mean tumor volume over time) of different groups are shown inFIGS. 24(a) and 24(b).

TABLE 8 Antitumor activity in the treatment ofNCI-H1975-L858R-T790M-C797S model RTV T/C Synergy RTV T/C Synergy @ @ratio @ @ ratio mRECIST Treatment D22 D22 @ D22 D29 D29 @ D29mRECIST_best @ D29 Vehicle 5.6 ± 0.6 — — 9.0 ± 1.3 — — 5/5 PD 5/5 PDcontrol Compound 15 3.4 ± 0.6 0.590 — 5.7 ± 1.7 0.630 — 5/5 PD 5/5 PDCisplatin 3.7 ± 0.9 0.780 — 4.6 ± 1.5 0.510 — 1/5 SD, 4/5 PD 5/5 PD TXT1.2 ± 0.3** 0.380 — 1.1 ± 0.2* 0.120 — 1/5 PR, 2/5 SD, 3/5 SD, 2/5 PD2/5 PD Compound 15 + 1.5 ± 0.4** 0.380 1.55 1.9 ± 0.7* 0.210 1.57 1/5PR, 1/5 SD, 1/5 SD, Cisplatin 2/5 PD 4/5 PD Compound 15 + 0.3 ±0.0**^(&) 0.120 2.53 0.4 ± 0.1* 0.050 1.65 5/5 PR 4/5 PR, TXT 1/5 SD *p< 0.05 vs control, ** p < 0.01 vs control; ^(&) p < 0.05 vs TXT; Ratio >1, Synergistic; Ratio = 1, Additive; Ratio < 1, Antagonistic

Example 11. Study on the Effect and the Potential Mechanism of Compound31 or Compound 15 on the Growth of Gastric Cancer Cells

(1) The sensitivity of gastric cancer cells to Compound 31 is closelyrelated to the expression of Bcl-2, Bcl-xL and Bax.

The inventors first analyzed the basal expression levels of Bcl-2 familyproteins in gastric cancer cell lines: AGS and NCI-N87 (purchased fromNanjing Cobioer Biotechnology Co., Ltd.), by Western blotting (FIG. 25).AGS and NCI-N87 are cell lines having high expression levels of Bcl-2and Bcl-xL and high expression level of Bax.

The inventors evaluated the inhibiting effect of Compound 31 (suppliedby Suzhou Ascentage Pharmaceutical) on the proliferation of gastriccancer cell lines by measuring the cell viability by CCK-8, and foundthat Compound 31 inhibited the proliferation of cell lines having highexpression levels of Bcl-2, Bcl-xL and Bax (AGS and NCI-N87) in aconcentration-dependent manner within 72 hours. The IC50s in AGS andNCI-N87 were 1.146±0.56 μM and 0.9007±0.23 μM, respectively (FIG. 26(a)and FIG. 26(b), Table 9). Thus, the sensitivity of gastric cancer cellsto Compound 31 is closely related to the expression of Bcl-2, Bcl-xL andBax.

TABLE 9 Cell line

  IC₅₀ (μM) 

AGS 

 1.146 ± 0.56 

N87 

0.9007 ± 0.23 

(2) Compound 31 had no significant effect on cell cycle.

The inventors used the cell cycle detection kit to perform cell cycleassays, and the results showed that Compound 31 had no significanteffect on cell cycle of both AGS and NCI-N87 gastric cancer cell lines(FIG. 27).

(3) Compound 15 significantly inhibited the proliferation of gastriccancer cells as the concentration increased in vivo.

In order to evaluate the anti-proliferative activity of Compound 15 invivo, the inventors constructed a NCI-N87 xenograft model using nudemice. The NCI-N87 tumor-bearing mice were treated with different dosesof Compound 15 by tail vein injection for 10 consecutive days. 5 weeksafter the inoculation of tumor cells, NCI-N87 tumor-bearing mice weresacrificed to remove the tumors for study. The measurement of tumorvolume found that Compound 15 as a single agent could exert asignificant effect on inhibiting tumor growth as the dose increased.Upon the end point, the mouse tumor volumes in the 25 mg/kg group andthe 50 mg/kg group grew to 700 mm³ and 800 mm³. The mouse tumor volumein the 100 mg/kg group was only about 400 mm³, while the control groupreached about 1300 mm³ (FIG. 28(a)). There were significant differencesamong these four groups.

In the tumor issues removed from the scarified mice, it was found thatas the dosing concentration increased, the weight and size of thexenograft tumors showed an evident trend of reduction (FIG. 28(b) andFIG. 28(c)). On the other hand, the measurement of mouse body weightshowed no significant difference in body weight among the four groups(FIG. 28(d)), suggesting that Compound 15 had a safe dosing window.

The inventors extracted proteins from the removed tumor tissues andprepared tissue sections. It could be seen that the protein expressionlevels of cleaved-caspase3 and PARP-1 cleavage bands in the tumor tissuegradually increased as the dose increased (FIG. 28(e)).Immunohistochemical staining showed that the percentage of Ki67-positivecells in the 100 mg/kg group was significantly lower than other groupsas the dosing concentration increased (FIG. 28(f)), suggesting theCompound 15 had inhibitory effect on the proliferation of gastric cancercells in vivo.

Example 12. Study on the Effect and the Potential Mechanism of aCombination of Compound 15 or Compound 31 with 5-Fluorouracil on theGrowth of Gastric Cancer Cells

(1) Compound 31 combined with 5-FU enhanced the induction of apoptosisin gastric cancer cells.

The inventors chose to combine Compound 31 with one of the most commonchemotherapeutic drugs in clinic, 5-fluorouracil (5-FU). In order toinvestigate the effect and the potential mechanisms of Compound 31 and5-FU on the growth of gastric cancer cells, the inventors used theAnnexinV/PI staining to detect the apoptosis of two gastric cancercells, AGS and NCI-N87, 48 hours after the treatment with Compound 31(0.3 μM), 5-FU (3 μM), and the combination at specified concentrations.In AGS cells, the treatment with Compound 31 or 5-FU as a single agentresulted in an apoptosis rates of approximately 22% or 15%, whereas inthe combination group, the proportion of apoptotic cells increased to54% (FIG. 29(a)). In NCI-N87 cells, the combination group also achieveda higher apoptosis rate than the mono therapy groups, Compound 31 or5-FU as a single agent leading to apoptosis rates of 24% or 18% whilethe combination group reaching an apoptosis rate of 46% (FIG. 29(a)).

At the same time, Western blotting showed that the cleaved-caspase3 andPARP-1 cleaved bands in AGS or NCI-N87 gastric cancer cells increasedmore significantly in the combination group than in eithermonotherapy-treated group (FIG. 29(b))). These results suggested thatboth in AGS and NCI-N87 gastric cancer cells, the combination ofCompound 31 and 5-FU enhanced the induction of apoptosis in gastriccancer cells.

(2) The combination of Compound 15 and 5-FU significantly inhibited theproliferation of gastric cancer cells by inducing apoptosis in vivo.

In order to evaluate the anti-proliferative activity of the combinationof Compound 15 and 5-FU in vivo, the inventors constructed a NCI-N87xenograft model using nude mice. The mice were divided into 4 groups:vehicle, Compound 15 (50 mg/kg qd), 5-FU (25 mg/kg) and the combination.Compound 15 was administered through tail vein injection for 10consecutive days, and 5-FU through tail vein injection once a week fortwo consecutive weeks. 5 weeks after the inoculation of the tumor cells,the mice were sacrificed to remove the tumors for study. The tumorvolume of the combination group was significantly smaller than the othergroups at any different measurement time. Upon the end point, the mousetumor volumes of Compound 15 and 5-FU monotherapy grew to 1600 mm³ and2200 mm³, and the tumor volume of combination group was only about 1000mm³ while the control group reached about 2600 mm³ (FIG. 30(a)). Therewere significant differences among these four groups.

Significant differences in the weight and size of the xanograft tumorsamong the combination group and the other groups were also found in thetumor tissues removed from the scarified mice, the combination groupbeing significantly smaller than the other groups (FIG. 30(b) and FIG.30(c)). On the other hand, the measurement of mouse body weight showedno significant difference among the four groups. Groups treated withCompound 15 and the combination rebounded after a brief weight loss(FIG. 30(d)), suggesting that Compound 15 had a good dosing window.

The inventors embedded the removed tumor tissues to prepare sections,and used TUNEL kit and immunohistochemistry assays for detection. TheTUNEL-positive cells in combination group were significantly more thanthe other groups (FIG. 30(e)). Compared with the other groups, theexpression of cleaved-caspase3 was also significantly increased in thecombination group (FIG. 30(f)), which all suggested that combination ofCompound 15 and 5-FU more effectively induced apoptosis in gastriccancer cells in vivo. The percentage of Ki67-positive cells in thecombination group was also significantly lower than that in the groupswith Compound 15 or 5-FU or the control group (FIG. 30(f)), suggestingthe more effective inhibiting effect of Compound 15 in combination with5-FU on proliferation of gastric cancer cells in vivo.

Example 13. Study on the Effect of Compound 31 in Combination withDocetaxel (TXT) on the Growth of Gastric Cancer Cells

Anti-proliferation effects were detected by CCK-8 (Cell Counting Kit-8)assay based on water soluble tetrazolium salt (WST). Briefly, NCI-N87cells were seeded in 96-well plates and treated with different Compound31 at different concentrations for 72 hours. To test the synergisticeffect between Compound 31 and docetaxel, docetaxel was added to eachwell at a concentration of 0.001 nM, 0.1 nM or 10 nM. Each concentrationwas prepared in triplicates. At the end of treatment, CCK-8 agents (10μL/well) were added to the 96-well plate, and incubated with the cellsfor 2 hours. The OD450 value was measured using a microplate reader. Thepercent cell viability was calculated using mean OD value of thetriplicate wells by the following formula:(O.D. test well−O.D. blank control well)/(O.D. cell control well−O.D.blank control well)×100%.

The IC₅₀ was calculated using the nonlinear regression data analysismethod of Graphpad Prism 6.0 software (Golden software, Golden, USA).The results are shown in FIG. 31.

The IC₅₀ value of Compound 31 in NCI-N87 cells was 0.405 μM as shown inFIG. 31. When cells were co-treated with docetaxel at a concentration of0.001 nM, 0.1 nM or 10 nM, the IC₅₀ values were reduced to 0.012, 0.014and 0.004 μM. A significant decrease in IC₅₀ values indicated thatCompound 31 and docetaxel had strong synergistic anti-proliferativeactivity in NCI-N87 cells.

Example 14. Study on the Effect of Compound 31 in Combination withPanobinostat on the Growth of Gastric Cancer Cells

Anti-proliferation effects were detected by CCK-8 (Cell Counting Kit-8)assay based on water soluble tetrazolium salt (WST). Briefly, HGC-27cells were seeded in 96-well plates and treated with Compound 31,panobinostat or the combination of Compound 31 and panobinostat atdifferent concentrations for 24 hours. At the end of treatment, CCK-8reagents (10 μL/well) were added to the 96-well plate, and incubatedwith the cells for 2 hours. The cell viability of each treatment groupunder each concentration was calculated, and the results are shown inFIG. 32.

As shown in FIG. 32, the treatment of cells with the combination ofCompound 31 and panobinostat at different concentrations resulted in amore significant decrease in cell viability as compared to the treatmentwith Compound 31 or panobinostat alone. The treatment with Compound 31in combination with panobinostat had a stronger anti-proliferativeactivity in HGC-27 cells.

What is claimed is:
 1. A method of treating a cancer in a patient inneed thereof, comprising administering to the subject a Bcl-2/Bcl-xLinhibitor, wherein the Bcl-2/Bcl-xL inhibitor is a compound having thefollowing formula

or a pharmaceutically acceptable salt thereof; wherein the cancer isselected from small cell lung cancer (SCLC), prostate cancer, ovariancancer, gastric cancer, pancreatic cancer, atypical thymoma, breastcancer, non-small cell lung cancer (NSCLC), melanoma; wherein thetreatment comprises administering the Bcl-2/Bcl-xL inhibitor on days 1,4, 8, 11, 15, 18 and 22 of the 28-day treatment cycle at a dose of about10 mg, 20 mg, 40 mg, 80 mg, 160 mg, 240 mg, 320 mg, or 400 mg; or thetreatment comprises administering the Bcl-2/Bcl-xL inhibitor on days 1,8, 15 and 22 of the 28-day treatment cycle at a dose of about 10 mg, 20mg, 40 mg, 80 mg, 160 mg, 240 mg, 320 mg, or 400 mg.
 2. The method ofclaim 1, wherein the patient is relapsed from or refractory to a priortreatment.
 3. The method of claim 2, wherein the prior treatmentcomprises surgery, chemotherapy, radio therapy, targeted therapy,immunotherapy, or any combination thereof.
 4. The method of claim 1,wherein the Bcl-2/Bcl-xL inhibitor is administered through intravenousinfusion.
 5. The method of claim 1, wherein the the cancer is SCLC. 6.The method of claim 1, wherein the prostate cancer is prostateadenocarcinoma.
 7. The method of claim 1, wherein the ovarian cancer isselected from epithelial ovarian tumor, ovarian germ cell tumor, andovarian stromal tumor.
 8. The method of claim 1, wherein the gastriccancer is gastric adenocarcinoma.
 9. The method of claim 1, wherein thepancreatic cancer is exocrine cancer or ampullary cancer.
 10. The methodof claim 1, wherein the breast cancer is selected from ductal carcinomain situ, invasive breast carcinoma, phyllodes tumor, and breastangiosarcoma.
 11. The method of claim 1, wherein the NSCLC is selectedfrom lung adenocarcinoma, squamous cell lung carcinoma, and large celllung carcinoma.